Book 2 Mobile Corona Diagnostic Lab Manual.pdf

Mobile Corona Diagnostic Lab Manual
Version 1.0
May 10th 2021
Authors: Susanne Pettinger, Leo Sprengel, Andreas Jefimowicz, Rüdiger Trojok
Contact:
Rüdiger Trojok
Director Venture Lab Molecular Assembly
trojok@unternehmertum.de
UnternehmerTUM GmbH
Lichtenbergstr. 6
85748 Garching, Germany
www.unternehmertum.de
www.utum-bio-kitchen.de
Facebook | Twitter | LinkedIn | Instagram
Managing Directors:
Prof. Dr. Helmut Schönenberger (CEO), Claudia Frey,
Stefan Drüssler, Dr. Andreas Liebl, Thomas Zeller
Chairwoman of the supervisory board: Susanne Klatten
Register Court: HRB 141703
VAT: DE 252 789 694

Content
Workshop 5
Table 1: Training for Mobile Lab LAMP Sars-Cov-2 Diagnostics 5
Getting started 6
Introduction 7
Coronaviruses 7
Figure 1: Sars-CoV-2 Introduction, Symptoms and Prevention 7
Room layout and workflow 8
Film 1: “Setting up the mobile Lab” 8
Figure 2: Workflow and room layout in a PCR laboratory. 9
The four mobile lab stations and rooms 9
Figure 3: Mobile Lab stations 10
Patient sampling room 10
Sample preparation room 10
Table 2: Extraction 1 station 11
Table 3: Extraction 2 station 11
Reagent preparation room 11
Table 4: Preparation station 12
Amplification room 12
Table 5: Amplification station 12
Application requirements for the mobile lab 13
Hygiene and safety 14
Table 6: Materials list for lab safety and hygiene products 14
General guidelines for sterile working 14
Film 2: “Hygiene concept” 14
Protocol 1: Hand washing 15
General guidelines for working with RNA 16
Protocol 2: Working with RNA 16
Working with SARS-CoV-2 17
Clothing and shoes 17
Further protective equipment 17
Protocol 3: Putting on and taking off personal protective equipment 18
Protocol 4: Cleaning and disposing personal protective equipment 19
Cleaning the Mobile Corona Diagnostic Lab 20
Table 8: Materials list for cleaning and disinfection routine 20
Protocol 5: General disinfection procedure using Dismozon Plus 21
Protocol 6: Handling spillages of samples 22
Protocol 7: Clean-up after a SARS-CoV-2 case among employees 23
Waste management 25
Figure 4. Overview of solid waste management during COVID-19 pandemic 25
Segregation 25
Table 9: Waste classification and handling instructions 26
Liquid waste 26
2

Autoclave 27
Protocol 8: Using the autoclave 27
Incineration 28
Diagnostic Methods 29
Patient data management 29
Patient Sampling 29
Sampling Safety and precautions 30
How to take an oropharyngeal swab sample 30
Table 10: Materials list for Patient sampling 32
Protocol 9: The following procedure should be performed for oropharyngeal swab sampling: 32
Sample Buffer and Storage 33
RNA extraction 34
Film 3: “RNA extraction” 34
Table 11: OXGEN Viral RNA Extraction Kit components and storage conditions 34
Table 12: Materials list for Viral RNA Extraction Supplied by User 34
Protocol 10: RNA extraction protocol from swabs or buccal swabs 35
Wash buffer preparation 35
RT-qPCR 36
Figure 5: RT-QPCR schematic 37
Film 6: “RT-qPCR setup” 37
RT-qPCR kit 37
Specifications and limitations 37
Table 13: Materials list RT-qPCR on extracted RNA samples 37
Protocol 11: Setting up an RT-qPCR run with extracted RNA 38
Running and analyzing an RT-qPCR run 39
Film 7: “qPCR run + analysis” 39
Protocol 12: Running the RT-qPCR assay 39
Figure 6: RT-QPCR result interpretation 40
RT-LAMP 41
Figure 7: RT-LAMP schematic 41
Film 4: “RT-LAMP assay setup” 42
RT-LAMP kit 42
Specifications and limitations 42
Table 14: Materials list for RT-LAMP with extracted RNA 42
Protocol 13: Setting up RT-LAMP reactions with extracted RNA 43
Running and analyzing an RT-LAMP assay 44
Film 5: “RT-LAMP run and analysis” 44
Protocol 14: Running and documenting the RT-LAMP assay 44
Figure 8: Schematic for interpretation of RT-LAMP results 45
Troubleshooting 45
Abbreviations 47
References 47
Appendix 47
3

Educational Videos 47
Film 1: “Setting up the mobile Lab” 47
Film 2: “Hygiene concept” 47
Film 3: “RNA extraction” 47
Film 4: “RT-LAMP assay setup” 47
Film 5: “RT-LAMP run + analysis” 47
Film 6: “RT-qPCR setup” 48
Film 7: “qPCR run + analysis” 48
Starter Pack Inventory 48
Table 15: Starter Pack Materials list for the mobile lab 48
Chemical Safety instructions 49
4

Workshop
Participants learn the necessary background information and techniques to diagnose
Sars-CoV-2 in our Mobile Corona Diagnostic Lab. The lab is shipped ready to use with all
devices, reagents, certificates and protocols. After one day of preparation together with a
local assistant, a five day course is given where every step is carried out by each participant.
The learning process is accompanied by UnternehmerTUM experts with written teaching
materials, video training, virtual live coaching and short exams to verify the learned content.
Reverse-Transcriptase Loop-Mediated Isothermal Amplification of
RNA (RT-LAMP)
Molecular Diagnostic Workshop
Date: 5th-9th July 2021
Venue: Department of
Molecular Medicine,
KSMD-KNUST
Time DAY 1: Opening Ceremony (5th
July, 2021) Remarks
09:00 10:00 Arrival and registration of participants Jasper?
10:00 10:15 Opening prayer
10:15 11:00 Welcome and Introduction
- Welcome Message: Prof. R. Ngala
- Introduction of Chairperson and invited guests
- Response from Chairperson
Prof. R. Ngala
Who?
Prof. R.C. Abaidoo
11.00 12.00 Project summary talk: mobile lab and LAMP diagnostics Rüdiger
12:00 13:30 LUNCH + GROUP PHOTO
13:30 14:00 Speed dating + group arrangements Jasper?
14:00 16:00 Lab 1 - session 1: Introduction to mobile lab Leo
Time DAY 2: Hygiene, RNAse-free working (6th
July, 2021) Remarks
09:00 10:30 Laboratory safety Leo
10:30 11:00 SNACK
11:00 12:30 hygiene and RNAse-free working Andi
12:30 13:30 LUNCH
13:30 15:00 Lab 2 - session 1 | group 1+2: Riboflavin and pipetting exercise
group 3+4: safety equipment + swab sampling
Rüdiger, Andi, Leo,
Sammy
15:00 16:30 Lab 2 - session 2 | group 1+2: safety equipment + swab sampling
group 3+4: Riboflavin and pipetting exercise
Sammy
5

Time DAY 3: Basics of molecular biology and RNA extraction (7th
July, 2021)
Remarks
09:00 10:30 Introduction to molecular biology and genetics Rüdiger, Lado
(online)
10:30 11:00 SNACK
11:00 12:30 RNA extraction theory Lado (online)
12:30 13:30 LUNCH
13:30 16:00 Lab 3 - session 1 | group 1+2+3+4: RNA extraction (+ evtl. Nanodrop?) Rüdiger, Andi, Leo
Time DAY 4: Diagnostic methods, RT-qPCR and RT-LAMP (8th
July,
2021)
Remarks
09:00 10:30 Diagnostic methods and data management Andi, Susanne
(online)
10:30 11:00 SNACK
11:00 12:30 The principles of PCR, RT-qPCR and RT-LAMP Susanne (online)
12:30 13:30 LUNCH
13:30 16:30 Lab 4 - session 1 | group 1+2: RT-qPCR + RT-LAMP
group 3+4: RT-qPCR + RT-LAMP
Sammy
16:30 17:30 Lab 4 - session 2 | evaluation of qPCR and LAMP Rüdiger, Andi, Leo,
Sammy
Time DAY 5: Data evaluation and wrap-up (9th
July, 2021) Remarks
09:00 10:30 Data analysis and diagnosis, repetition of diagnostics Rüdiger, Andi, Leo
10:30 11:00 SNACK
11:00 12:30 Lab 5 - session 1 | group 1+2: troubleshooting + repetition
group 3+4: LAMP Primer design
Sammy, Jasper
12:30 13:30 LUNCH
13:30 15:00 Lab 6 - session 1 | group 1: LAMP Primer design
group 2: troubleshooting + repetition
Sammy, Jasper
15:00 16:00 feedback and plenary discussion Sammy, Jasper
16:00 17:00 presentation of certificates Sammy, Jasper
6

Getting started
1. Carefully read this book and make yourself familiar with the described methods.
2. Set up your Mobile Corona Diagnostic Lab as described in chapter x.
3. Equip the Mobile Lab as described in chapter x and check all received equipment
and machines as described in chapter x.
4. Make yourself familiar with the basic hygiene protocols described in the Hygiene and
safety regulations.
5. Perform a routine cleaning protocol as described in the Protocol: General disinfection
procedure using Dismozon Plus before you start working.
6. Every morning or before starting a work shift, obtain swab samples from all staff
working in the Mobile Lab as described in the Protocol: How to take an
oropharyngeal swab sample.
7. Perform RNA extraction on obtained swabs as described in RNA Extraction Protocol
from swab/buccal swab.
8. Set up and run an RT-LAMP assay as described in the Protocol: Setting up RT-LAMP
reactions with extracted RNA.
9. Optionally, set up and run an RT-qPCR assay as described in the Protocol: Setting
up an RT-qPCR run with extracted RNA.
10. Analyse your staff samples as described in the chapter Analyzing and interpreting an
RT-LAMP assay (RT-LAMP) and Analyzing and interpreting an RT-qPCR run
(RT-qPCR) to ensure they are SARS-CoV-2 negative. If one of your staff was tested
SARS-CoV-2 positive, refer to the Protocol: Clean-up after a SARS-CoV-2 case
among employees.
11. When all staff was tested SARS-CoV-2 negative, you can start operating the Mobile
Lab by obtaining patient swab samples.
7

Introduction
Coronaviruses
The novel SARS-CoV-2 virus belongs to the family of Coronaviridae, which are known to
cause several upper respiratory tract infections in humans. These viruses are encased by a
viral envelope and contain single-stranded RNA molecules carrying their genetic information.
SARS-CoV-2 particles are spread via small droplets which are expelled when an infected
person coughs or sneezes, but they can also be contained in aerosols expelled with
breathing. The most common initial symptoms of a SARS-CoV-2 infection are fever,
coughing and a shortness of breath. A severe course of disease can also lead to
pneumonia. As individuals mainly infect themselves when they inhale or ingest viral particles
contained in droplets or aerosoles, some simple measures can help protect against
SARS-CoV-2 infection. A strict personal hygiene with regular hand washing, routine
disinfection of surface, social distancing and strict quarantaines of infected persons can help
to limit the spread of SARS-CoV-2 effectively (Figure1).
Figure 1: Sars-CoV-2 Introduction, Symptoms and Prevention
Coronaviruses can cause a number of acute and chronic diseases causing upper respiratory
tract infections, bronchiolitis, and pneumonia in humans. The novel coronavirus
SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), was discovered in 2019
due to a cluster of pneumonia cases in Wuhan City, China (Sohrabi et al. 2020) (Gorbalenya
et al. 2020). It causes a disease named COVID-19 (coronavirus disease 2019) with
symptoms including pneumonia, fever, dry cough, fatigue, chest pain and myalgia (Huang et
al. 2020; Harapan et al. 2020). On March 11, 2020 the World Health Organization (WHO)
precleared a global pandemic. Early detection and isolation of infected patients are key to
control the spread of SARS-CoV-2 and disrupt the chain of transmission. In response to the
fast worldwide spread of SARS-CoV-2, the Mobile Corona Diagnostic Lab has been
developed as a Point-Of-Care (POC) solution for flexible Covid-19 diagnostics. It consists of
8

three subunits and combines sample collection and RNA extraction with sensitive genomic
detection via RT-LAMP and RT-qPCR.
This document includes a complete concept for patient sample management, guidelines for
handling infectious material, instructions for personal hygiene to ensure health and safety of
personnel, and guidelines to avoid contaminations with nonspecific RNA or RNAses which
could interfere with the reliable detection of SARS-CoV-2.
Room layout and workflow
For Covid-19 Diagnostics, and PCR laboratories in general, it is important to separate the
infectious patient samples from the reagents preparation and the amplification products to
avoid cross contamination via aerosols and liquid spillage. Thus, a testing laboratory has to
have a least 4 rooms with separate entrances for each room: a patient sampling room where
patients samples are taken with a swab, a sample preparation room, where the swabs get
processed to isolate RNA from putative virus infections, a reagent preparation room, where
RNA isolates and PCR master mixes get set up and an amplification room, where the
pathogenic RNA is detected by PCR or LAMP nucleic acid amplification methods to provide
a patient diagnosis. Each room has its dedicated set of materials and equipment, which
stays in the room at all times. Per room, one technician is employed and there should be no
moving in between areas to reduce risk of carryover contaminations. The patient sampling
room and the sample preparation room are in contact with infectious patient samples and
are thus in the biosafety category S2 and require special hygiene protocols to be followed
(see chapter Hygiene and Safety). The reagent preparation and amplification rooms are
regular biosafety level 1 labs.
Film 1: “Setting up the mobile Lab”
The only materials which move between the rooms are the patient samples from the
sampling room to the preparation room, and after RNA Extraction the virus RNA isolates
from the patient sampling room into the reagent preparation room. From there, the prepared
PCR and LAMP samples are handed over to the amplification room. This “unidirectional
flow” of material ensures that no cross contamination can occur across the workflow. It also
protects the technicians from infectious viral material, as the contact is limited to the patient
sampling and sample preparation rooms. After amplification, samples have to remain
unopened and need to be handled with care and must never be passed back to the other
rooms, due to high amounts of pathogenic nucleic acids which could in case of spillage and
cross contamination result in false positive results (Figure 2).
9

Figure 2: Workflow and room layout in a PCR laboratory.
S2 Patient sampling room for taking a swab from the mouth and nose of a patient (patient registration happens
in a different location prior to sampling). S2 Sample Preparation Room for processing of sample, extraction of
nucleic acids and addition of patient sample to master mix aliquot. S1 Reagent Preparation room for PCR
master mix preparation and aliquoting and adding of patient RNA isolates. S1 Amplification room for real-time
reverse transcription PCR with digital readout and reverse transcription LAMP with photo documentation.
Arrows indicated entrance and exit of patient and unidirectional workflow across the rooms. Each room has a
dedicated mobile lab infrastructure and set of equipment.
The organisation which operates the mobile lab besides these four lab rooms further needs
to provide a storage room for consumables and hygiene articles and also needs to have an
incinerator on the premise for waste disposal. Further, the patient management in the facility
with patient registration as well as presentation of the diagnosis has to be arranged
according to local circumstances and technical infrastructure available and are not covered
in this handbook.
The four mobile lab stations and rooms
The Mobile Corona Diagnostic Lab consists of four modules (Figure3), two for the sample
preparation room and one each for reagent preparation and amplification rooms. There is no
dedicated station for the patient sampling room, where a table and a chair for the patient
should suffice. The following chapter provides a description of each station and resents a
system for storing the items needed in the testing process.
10

Figure 3: Mobile Lab stations
Every station has drawers, each including two sturdy plastic storage boxes. Drawers are labelles and the
stations are color coded for easier identification. Red for Extraction 1 and 2 for the patient sampling room,
yellow is assigned for the Preparation station and green for Amplification station and for their matching rooms.
Inside each drawer is space for two flat or medium high Eurocontainers. (pictures may vary slightly from the
actual furniture layout)
Patient sampling room
In the Patient sampling room the oral and nasopharyngeal samples are collected from the
patients by a trained medical technician. The room has to be cleaned and disinfected
regularly due to patient contact which brings in potential S2 pathogens such as corona
viruses (see hygiene concept). Staff must wear full personal protective equipment at all
times (full-body protective suit, FFP2/FFP3 mask, safety goggles, gloves). It is important to
test only one patient at a time. Patients enter from one door and leave by another to avoid
contact and crowding of patients. Before testing, the sample is documented with a patient
specific ID assigned and labeled and the sample forwarded to the Sample preparation room
for further procedure (see patient data management chapter).
Sample preparation room
The sample preparation room (red) contains the modules: Extraction 1 and Extraction 2.
The Stations are connected to each other with a connection table element. Extraction 1
provides a sink for handwashing and discarding non contagious fluids as well as items for
autoclaving and other hygiene items. Extraction 2 provides all equipment and items needed
to perform the RNA extraction protocol (see RNA extraction).
The stations are used to process incoming swab-samples and isolate RNA-samples.
Qualified lab technicians with sufficient training are required to perform the extraction
protocol. The staff is assigned in 4h hour shifts and does not move to other working areas
meanwhile. They must wear at all times full personal protective equipment (full-body
protective suit, FFP2/FFP3 mask, safety goggles, gloves). The Room must be cleaned and
disinfected regularly to keep it free of RNases and infectious viral particles (See Hygiene).
After processing, lab trash including the swabs and consumables get autoclaved and
discarded (see waste management chapter). Only sterile tubes labelled with patient specific
IDs with the RNA-sample inside are allowed to leave the room without autoclaving.
11

Table 2: Extraction 1 station
Drawer 1 (sink) Drawer 2 (small) Drawer 3 (small) Drawer 4 (big)
Box 1 Box 2 Box 1 Box 2 Box 1 Box 2
SINK
soap dismozon
overalls
autoclave
tape
centrifuge storage during
transport
Autoclave storage during
transport
skin
disinfectant RNAse erase
autoclave
bags big
gloves FFP2 masks
hand balm duct tape safety goggles
Hand wash
station 2 wash bottles
Table 3: Extraction 2 station
Drawer 1 (big) Drawer 2 (big) Gap 3 Drawer 4 (small) Drawer 5 (small)
Box 1 Box 2 Box 1 Box 2 fridge Box 1 Box 2 Box 1 Box 2
pipette
holder pipette tips 10µl
eppis 1.5 ml table-top
waste
patient
sample
storage
eppendorf
racks
falcon racks RNA
extraction
kits
gloves
pipette 10 µl pipette tips 100
µl
eppis 2.0 ml waste bags
pipette 100
µl
pipette tips
1000µl
vortexer
pipette 1000
µl label printer
labels
tissue
Equipment which comes with this station are:
Autoclave: used to sterilize possibly contaminated working materials and patient samples to
a germ density of almost zero by heat and overpressure. Handle with care and study
manufacturer documentation before use.
UV-Lamp (optional): works in an overnight mode to decontaminate working surfaces.
Pipette-UV-Holder with 10,100 and 1000µl pipetttes
Centrifuge model Hermle Z216-M-E with cooling function for RNA-Extraction-Spin-Columns
Trashbin (not provided): trash bin for autoclave waste - have to be equipped with
autoclaving bags.
fridge: for temporary storage of patients samples until the next RNA extraction run.
Reagent preparation room
The reagent room (yellow) contains the mobile lab station ‘Preparation’. The Preparation
station is used to store and prepare reagents such as the amplification master mixes of
RT-LAMP and RT-qPCR kits, aliquoting and preparation of positive and negative controls
and adding extracted RNA from patient sample to the aliquots. (see chapters on RT-qPCR
and LAMP). The station has a dedicated set of adjustable pipettes with plugged,
aerosol-barrier tips, laboratory coats and disposable gloves. The room is a S1 level facility
12

and staff has to wear basic personal protective equipment (lab coat, FFP2 mask, gloves,
goggles). The station has to be cleaned and disinfected regularly to keep free of RNases
and contaminating external RNA (see hygiene concept). The technician in this room receives
the RNA isolates from the sample preparation room. To prevent cross contamination and to
avoid repeated freezing and thawing, reagent-stock solutions should be aliquoted into
smaller volumes before processing a batch of extracted RNA patient samples. The
technician then prepares the master mixes while making sure that by adequate sample
labeling, no patient samples get confused or swapped accidentally. Special care has to be
taken to not spill any sample and to avoid cross contamination and carryover problems.
Trashbin (not provided): trash bin for incineration waste. Need be be equipped with waste
bags.
Freezer (not provided): Enzymes for the PCR and LAMP Reactions are stored in the freezer
to make sure that they are prevented from denaturation/inactivation.
Table 4: Preparation station
Drawer 1 (big) Drawer 2 (big) Drawer 3 (small)
Drawer 4
(small)
Drawer 5
(small)
Drawer 6 (small)
Box 1 Box 2 Box 1 Box 2 Box 1 Box 2 Box 1 Box 2 Box 1 Box 2 Box 1 Box 2
pipette
holder
pipette
tips 10µl
lab
coats
vortexe
r gloves
S/M/XL
PCR tube
rack
PCR
tubes
nucleas
e-free
water
first aid
kit
Eye
wash
Dismoz
on waste
bags 60L
pipette
10 µl
pipette
tips 100
µl
safety
goggles tissue
wipes
metal
cooling
block
RNAse
Erase
table-top
waste
pipette
100 µl
pipette
tips
1000µl
FFP2
masks 2 wash
bottles
waste
bags
pipette
1000 µl
multi-ch
annel
Amplification room
This station uses the amplification module.
The Amplification Room is designated for the RT-LAMP/RT-qPCR run and analysis of
results. The thermoblock and real-time PCR machine are be kept in this room. The room is
S1 facility and the staff wears basic personal protective equipment (lab coat, FFP2 mask,
gloves). It is important that no vial is opened after amplification and a clean environment is
preserved. Cleaning of the machines must be done daily (See hygiene).
Table 5: Amplification station
Drawer 1 (big) Drawer 2 (small) Drawer 3 (small)
13

Box 1 Box 2 Box 1 Box 2 Box 1 Box 2
qPCR machine thermoblock gloves safety goggles wash bottles waste bags 60L
tablet FFP2 masks Dismozon tissue wipes
Trashbin (not provided): trash bin for incineration waste. Need to be equipped with waste
bags.
heat block: The heat block is used to perform the RT-LAMP-Reaction, if a sample is tested
as positive a qPCR is carried out in addition to confirm the result.
thermocycler chai RT-qPCR: Is used to perform a quantitative
Polymerase-Chain-Reaction. See machine user guide and chapter on RT-qPCR for more
information
tablet: used for documenting the results by storing the readouts of the QPCR and by taking
photos of the RT-LAMP reaction tubes together with the patient ID labels
Application requirements for the mobile lab
The mobile lab for Covid-19 diagnostics is a transportable set of laboratory furniture which is
equipped with all required equipment for QPCR and LAMP diagnostics. The requirements on
the preexisting infrastructure are kept to a minimum. The mobile lab can be employed in
places that are easily accessible by infected people. Neuralgic points such as hospitals,
schools, or improvised test tents, are example application areas.
For future uses, not only SARS-CoV-2 can be diagnosed, but all other diseases for which
there are either certified QPCR or LAMP reagents are available can be tested for.
Also it is possible to use the mobile lab in other medical applications or areas such as
forensics or veterinary applications.
What the operator of a mobile lab in any facility has to provide is the above mentioned rooms
with appropriate ventilation and access management. In order to avoid deviations in the test
results, the laboratory rooms should be air-conditioned and operated at a temperature range
of 20-25 degrees Celsius. Electricity from the grid needs a voltage to operate the integrated
devices of 230 volts with a frequency of 50 Hertz, which is the case for most of European
countries and also for Ghana. In case of blackout danger, an adequate power generator or
batteries need to be provided as backup solutions. For Ghana there are two associated plug
types, D and G. However, the G type is very common in Ghana. Plug type D is the plug
which has three round pins in a triangular pattern and type G is the plug which has three
rectangular pins in a triangular pattern. Which means adapters from German to Ghanaian
plugs are needed. Adapters are only required in the sockets of the premises, as European
multiple plugs are included with the Mobile Lab. Furthermore, a generously calculated supply
of fully demineralized water and ethanol must be available in order to ensure the operation of
the laboratory and the corresponding hygiene measures. For safe waste disposal, an
incinerator needs to be accessible on a daily basis. The operator also needs to make sure
that starter pack materials can be shipped at least once per week whereby a constant
cooling chain for the supply delivery is mandatory.
14

Hygiene and safety
It has to be assumed that every patient visiting the Mobile Corona Diagnostic Lab has a
SARS-CoV-2 infection until diagnosed otherwise. Wearing proper personal protective
equipment is therefore key to protect employees and health workers from SARS-CoV-2. In
addition, proper hand hygiene is a simple but effective way to prevent the spread of
pathogens such as SARS-CoV-2. Hands always have to be washed when visibly soiled, as
well as before and after eating or using the restroom. To prevent the spread of SARS-CoV-2,
hands should also be washed before and after touching your eyes, nose, mouth or face
mask, when entering or leaving a public place or after touching critical surfaces such as door
knobs, shopping carts or tables.
Table 6: Materials list for lab safety and hygiene products
Personal protective equipment Included in starter pack
Full-body protective suit DuPont Model Tyvek
Lab coat, cotton - -
gloves, nitril Noba 90595
safety goggles Carl Roth Y254.1
FFP2 mask Carl Roth EHT9.1
Shoes or protective plastic overshoes - -
duct tape Spada 52018745452012
Equipment Included in starter pack
waste bin 60 liters Tork 53 6664
autoclave bag for waste bin Carl Roth ATY4.1
UV-lamp - -
Chemicals and reagents Included in starter pack
Skin cleaning agent Herwe 10121605
Skin disinfectant agent Herwe 10203590
General guidelines for sterile working
Using sterile (aseptic) techniques during work is important to prevent contamination with
microorganisms and pathogens and to reduce the risk of infections when working with
patients. The four main aspects of sterile working, which you should follow as a general
rule, are explained below. Keep in mind: Work carefully and fast, but without any hurry!
Film 2: “Hygiene concept”
Barriers: Protect your samples from yourself
● use clean, appropriate personal protective equipment (PPE)
● wash and disinfect your hands before and after working, tie back long hair
● avoid speaking, singing and whistling while working
● if you have to sneeze or cough, direct yourself away from the work area
15

Equipment and preparation: Use high-quality, sterile equipment
● clean and disinfect equipment before and after use with 70 % (v/v) ethanol
● use sterile, disposable items where possible and immediately discard them after use
● only unwrap sterile items directly before using them
● avoid laying sterile equipment down on surfaces if possible, if you have to put down a
cap or lid place it with the opening facing down on a clean surface
● avoid pipetting chemicals directly from large bottles, instead pour yourself a sufficient
aliquot before you start working and discard it afterwards
Environmental controls: Maintain a sterile working environment
● keep the number of staff present as low as possible
● keep doors and windows closed to reduce air draughts and prevent sudden
movements to avoid disturbing the air
● ensure you have all required materials and equipment at hand before you start
working, remove all unnecessary equipment to keep your work area tidy
● disinfect the whole work area using 70 % (v/v) ethanol before you start working
● only open bottles and reaction tubes directly before you need them, do not leave
them open but close them again immediately after use to prevent airborne
contaminations
● if you spill something, clean up immediately and discard your gloves afterwards
Contact guidelines: How you should behave when handling delicate samples
● wear your PPE when touching sterile items
● only touch what you have to touch while working, absolutely avoid touching
non-sterile items and surfaces
● disinfect your gloves with 70 % (v/v) ethanol before you take equipment or after you
have touched non-sterile items or surfaces
● pay attention not to touch your face or hair while working, if you do so disinfect your
gloves with 70 % (v/v) ethanol before you continue
● do not touch the lower part of your pipette or the pipette tip
Protocol 1: Hand washing
Washing hands mechanically for 20 seconds removes pathogens and germs by enclosing them in foam
bubbles. The protocol for hand washing recommended by the WHO [ref] is very simple to follow:
● Remove any rings, wrist-watches or bracelets.
● Wet your hands with clean, running water.
● Apply soap and thoroughly rub your hands together to create foam.
● Scrub your hands for 20 seconds spreading the foam everywhere. Remember to also scrub the back
of your hands, between your fingers and under your nails.
● Rinse your hands well under clean, running water.
● Dry your hands using a clean towel or an air dryer.
If required, you can use a disinfectant after your hands are completely dry. Apply a palmful of disinfectant
and cover all surfaces of your hands with it. Rub your hands until they are dry.
16

General guidelines for working with RNA
RNA is rather difficult to work with for two reasons: It is a chemically instable molecule
susceptible to degradation by hydrolysis, and it can be degraded by ubiquitous enzymes
called RNAses. Ribonucleases (RNAses) are very stable enzymes which actively degrade
RNA. They are in general very robust enzymes which do not require cofactors to function
and can even remain active after prolonged boiling or autoclaving. This makes RNAses very
difficult to inactivate. As even minute amounts are sufficient to destroy any RNA in your
sample, you have to take great care not to introduce any RNAses into your samples or
reactions at any time point. Hands and dust particles can carry bacteria or molds and are the
most common sources of RNAse contamination. In order to protect your RNA samples and
to create and maintain an RNAse-free environment, take the following precautions in
addition to following the general guidelines for sterile working.
Protocol 2: Working with RNA
● always keep samples containing RNA on ice to prevent degradation as RNA is very sensitive to
elevated temperatures
● pay attention not to warm the samples up while holding them in your hand
● store RNA samples in small aliquots at -80 °C and prevent repeated freeze-thawing
● have a separate area designated for RNA work only and make sure that only properly trained
colleagues are working there
● clean all surfaces with an RNAse-removing agent directly before you start working and wipe them
again with 100 % ethanol
● clean all required equipment such as pipettes with an RNAse-removing agent before you use them
● always wear gloves to prevent RNAse contaminations from your skin and change gloves frequently,
especially after touching a non-sterile surface or item
● use sterile, RNAse-free disposables and do not use them more than once
● use pipette tips containing an aerosol barrier (filter)
● when you treat glassware before usage, keep in mind that autoclaving will not fully inactivate
RNAses
○ clean glassware with a detergent, rinse thoroughly, and bake it in an oven at 240 °C
overnight or
○ fill it with 0.1 % DEPC, allow to stand overnight at 37 °C and then autoclave
● use RNAse-free chemicals and store them separated from other reagents in the lab
● you can add RNAse inhibitors to protect your RNA from degradation during isolation and
purification
In addition to the general recommendations mentioned above, you can also use an UV-lamp
for ultraviolet germicidal irradiation, a process which will kill or inactivate microorganisms as
well as RNAses. UV light can be used to disinfect equipment including pipettes and your
safety goggles as well as for the sterilization of glassware. It can also be used to disinfect
surfaces or as a pre-treatment for SARS-CoV-2 contaminated waste. The effectiveness of
this method depends on the time of direct exposure, any obstacles blocking the UV radiation
can dampen the effect.
Be aware that UV-C light is hazardous also to humans, it can cause sunburn and skin
cancer. Wear protective glasses blocking UV-C light and do not look directly into the lamp.
Do not expose yourself to the lamp's UV irradiation.
17

Working with SARS-CoV-2
To prevent a spread of SARS-CoV-2 among health workers and lab employees, a simple but
strict hygiene and safety concept is recommended. The key points for infection prevention
and control are:
● frequent hand washing or disinfection with an alcohol-based hand rub
● respiratory hygiene, e.g. covering coughing or sneezing, avoid talking too much
● wearing masks and personal protective equipment
● physical distancing of at least 1.5 meter when working together
● regular cleaning and disinfection of the environment, including objects such as door
knobs, telephones and keyboards
In addition, employees of the Mobile Corona Diagnostic Lab should test themselves for
SARS-CoV-2 infection at the beginning of each work shift to protect colleagues as well as
patients.
Clothing and shoes
The full-body protective suits provided with the Mobile Corona Diagnostic Lab must be worn
by all employees directly exposed to viral particles. In particular, these overalls are
mandatory when taking patient swab samples or performing RNA extraction on samples. For
safety and hygiene reasons, full-body protective suits should not be swapped between
personnel unless they have been autoclaved before. If more than one person wears an
overall at the same time, it is recommended to label the overalls with the respective names
to prevent accidental swaps. It has to be assumed that the full-body protective suits are
contaminated after one day of use. If possible, these overalls should be autoclaved at the
end of each work shift before using them again. If the overalls are single-use items, they
have to be autoclaved before discarding them.
For employees working only with the RT-LAMP or RT-qPCR assays, a lab coat can be
sufficient as full-body protective suits are relatively expensive. However, employees wearing
only a lab coat are not allowed to enter the patient treatment room or the RNA extraction
area for their own safety. Lab coats should not be shared among employees, it is therefore
recommended to label them with names. Lab coats should be washed regularly, at least
once per week. If they are dirty or soiled, lab coats have to be washed immediately. Before
washing them, the lab coats have to be autoclaved for safety reasons.
It is recommended to use shoes that are only worn in the laboratory area. These shoes
should be comfortable, fully closed, wipeable and washable, and should not be worn outside
the laboratory. Alternatively, disposable plastic overshoes can be used. In this case, the
overshoes have to be put on when entering the patient treatment room or RNA handling
area and thrashed when leaving these areas.
Further protective equipment
The supplied protective masks have to be worn by every employee in the laboratory at all
times. Only wear FFP2 or FFP3 masks. Surgical masks or (self-made) fabric masks are not
suitable for use when working in the Mobile Corona Diagnostic Lab. Other face coverings
such as scarves or bandanas are also not acceptable. Protective masks must not be shared
among employees. It is therefore recommended to label them on the outside to prevent
18

accidental swaps. For hygiene reasons, masks should not be placed on surfaces. When they
have to be taken off, masks can be hung on a clothes hook. Protective masks should only be
touched after washing and disinfecting hands. They should not be touched on the inside,
which is in contact with the face. Protective masks are single-use items and should be
disposed of at the end of a shift. If the protective mask is dirty or wet, it has to be discarded
immediately. In case of shortages, clean protective masks can be dried on a clothes hook for
7 days before using them again [ref].
All employees working in the patient treatment room or the RNA extraction area must wear
safety goggles to protect them from SARS-CoV-2 virus particles.
All employees have to wear single-use nitrile or latex gloves. While the SARS-CoV-2
pandemic led to a global shortage of gloves, they are considered to be disposable items and
should be changed frequently if necessary. For hygiene reasons, always change gloves after
taking a swab sample from a patient. In addition, gloves should be changed if soiled or
damaged.
Protocol 3: Putting on and taking off personal protective equipment
Personal protective equipment (PPE) has to be put on and taken off in a specific order to ensure it does not
get contaminated or damaged and to protect yourself from self-contamination after work.
Employees taking swab samples from patients or running RNA extractions on patient material can be directly
exposed to SARS-CoV-2 virus particles. These employees must wear a full set personal protective equipment
consisting of an overall, a FFP2 face mask, safety goggles as well as gloves. Employees preparing, running or
analysing RT-LAMP or RT-qPCR assays have a lesser infection risk as they do not handle viral particles. In this
case, wearing a lab coat instead of an overall is acceptable.
See Video https://www.cdc.gov/coronavirus/2019-ncov/hcp/using-ppe.html
How to PUT ON personal protective equipment
PPE has to be put on correctly before entering the patient treatment room or RNA extraction area (that is,
before having patient contact). It has to be worn for the entire work time and should not be adjusted during
patient care. The sequence for putting on personal protective equipment is as follows:
● Obtain all necessary equipment, e.g. a sealed full-body protective suit, a packaged face mask,
gloves and safety goggles.
● Tie back long hair.
● Wash your hands with soap and disinfect them afterwards.
● Put on your work shoes or plastic overshoes.
● Put on the overall. Close it completely and seal the connection between pants and shoes with tape.
Check that the overall does not have holes or tears.
● Put on your FFP2 or FFP3 face mask. Adjust it to sit correctly and ensure the ribbons holding it in
place are adjusted.
19

● Put on your safety goggles. Adjust them so they sit comfortably without interfering with your face
mask.
● Put on a pair of gloves and pull them above the overall wrists. Seal the connection between gloves
and overall with tape.
How to TAKE OFF personal protective equipment
PPE should be removed carefully after work to prevent self-contamination. It should only be removed after
all patients have left the room. Leave the patient treatment room or RNA extraction area directly after
disposing your PPE. The sequence for taking off personal protective equipment is as follows:
● Remove your gloves, but be aware that the outsides of your gloves are contaminated!
○ Tear off the sealing tape around your wrists.
○ Carefully peel off the first glove holding it in your still-gloved hand.
○ Slide the fingers of your now ungloved hand under the wrist of the remaining second glove
and peel it off, turning the inside out and covering the first glove you are still holding.
○ Discard the gloves in the appropriate waste bin.
● Remove your goggles by lifting the band from the back or side of your head. Do not touch the front
or rim of your goggles as they can be contaminated.
● Remove your overall, but keep in mind that its front and sleeves are contaminated!
○ Unfasten the ties or open the zipper.
○ Peel off the overall from your neck and shoulders. Only touch it on the inside!
○ Turn the overall inside out while pulling it down to your feet. Step out of it.
○ Roll the overall into a bundle with the inside out and discard it properly.
● Take off your shoes or plastic overshoes. Avoid touching them as far as possible.
● Wash your hands with soap and disinfect them afterwards.
Protocol 4: Cleaning and disposing personal protective equipment
Apart from your safety goggles, all personal protective equipment has to be autoclaved first as it is
considered contaminated. Gloves and FFP2 face masks are single-use items and can be discarded after
autoclaving according to local regulations. For full-body overalls check the manufacturer and brand to
determine whether they can be reused after autoclaving, if not dispose of them according to local
regulations. Cotton lab coats can be washed after they have been autoclaved and reused.
Safety goggles are not autoclaved but cleaned and disinfected as followed:
● Gently rub the inside and outside with mild soap under clean running water.
● Rinse well with clean water to completely remove the soap and let the goggles dry.
● Wet the inside and outside with disinfectant and let it sit for at least 1 minute.
● Rinse again with clean water.
● Let your goggles dry completely before putting them on again.
20

Cleaning the Mobile Corona Diagnostic Lab
Apart from personal hygiene measures, cleaning and disinfecting all work areas are key for
effective infection control. In general, 0.8 % Dismozon Plus should be used for routine
cleaning every morning and evening or before closing the lab. Additional disinfection has to
be done if a contamination occurs (e.g. a sample is spilled) or if an employee has been
tested SARS-CoV-2 positive.
In areas where RNA is handled, surfaces and equipment should be treated with RNAse
Erase before work. In addition, all surfaces should be wiped with 70 % (v/v) ethanol
regularly. Ethanol is a cheap and very effective disinfectant, so you can use it generously to
wipe the area you are currently working in and use it to disinfect your gloved hands
whenever you change equipment or progress from one step in a protocol to the next.
Table 8: Materials list for cleaning and disinfection routine
lab coat, cotton - -
shoes or protective plastic overshoes - -
waste bin 60 liters Tork 53 6664
autoclave bag for waste bin Carl Roth ATY4.1
cleaning bucket - -
clean rag - -
measuring cylinder, 2 liters - -
autoclave Wisconsin 94638
Dismozon Plus Bode 981257
ethanol, diluted to 70 % (v/v) Carl Roth T913.3
RNase Erase MP Biomedicals 04821682
Skin cleaning agent Herwe 10121605
Skin disinfectant agent Herwe 10203590
clean water - -
21

Protocol 5: General disinfection procedure using Dismozon Plus
Dismozon Plus is an aggressive reagent which can cause skin burns and eye damage. It is recommended to
briefly open a window or the door while cleaning as it has a strong smell. After preparing the Dismozon Plus
solution it has to be used within 8 hours to guarantee the active oxygen´s antimicrobial effect.
1. In the morning, put on your personal protective equipment including a lab coat.
2. Prepare a solution of 0.8 % Dismozon Plus by adding one package (16 grams) to 2 liters of clean
water. Make sure it dissolves completely.
3. Use a clean rag to thoroughly wet all surfaces as well as the fronts of all cupboards with the 0.8 %
Dismozon solution. Wait 15 minutes for the solution to take effect.
4. Rinse the rag and use it to wipe all treated surfaces and fronts with clean water until no residual
Dismozon solution is left.
5. Wipe all surfaces and fronts with 70 % (v/v) ethanol and let them dry briefly.
6. Prepare your individual work area directly before you start working:
a. In the patient treatment room, use 70 % (v/v) ethanol to disinfect all surfaces which were
touched by patients, e.g. door knobs or arm chairs, after every patient to prevent
cross-infections among patients.
b. In the RNA extraction area, wipe the surface of the working area and all equipment with
RNase Erase and wipe again with 70 % (v/v) ethanol.
c. In the RT-LAMP and RT-qPCR preparation area, wipe the surface of the working area and
all equipment with RNase Erase and wipe again with 70 % (v/v) ethanol. While handling
patient samples and after handling the positive control, wipe the surface of the work area
with 70 % (v/v) ethanol and change your gloves afterwards.
d. In the assay run area, clean all machines with RNAse Erase and 70 % (v/v) ethanol before
you use them. Pay attention to also clean the inside of the instrument lids.
7. In the evening, use a rag to wipe the floor with the 0.8 % Dismozon Plus solution and wait 15
minutes for the solution to take effect.
8. Thoroughly rinse the rag and wipe the floor with clean water to remove all excess Dismozon
solution. Wait until the floor is completely dry before you enter again.
22

Protocol 6: Handling spillages of samples
If you spill a sample in the RNA extraction area, either before or after the patient material was inactivated,
the following protocol applies:
1. Soak up all spilled liquid using paper towels and immediately clean the spot with a generous
amount of 0.8 % Dismozon Plus solution followed by 70 % (v/v) ethanol.
2. If the spilled sample was extracted RNA obtained AFTER viral inactivation:
a. Disinfect the area around the spill as well as all machines and equipment in the room with
0.8 % Dismozon Plus solution and wipe again with clean water.
b. Clean all treated areas with 70 % (v/v) ethanol.
c. If your full-body protective suit or any of your personal protective equipment was soiled,
you have to change it.
d. Change your gloves and continue working.
3. If the spilled sample was patient material BEFORE viral inactivation, you have to disinfect the whole
RNA extraction area by running the full general cleaning and disinfection protocol.
If a PCR tube lid pops open in the assay run area after the reaction has finished, but no liquid has spilled,
apply the following protocol:
1. Immediately close the tube again and seal it tightly.
2. Disinfect the area, equipment and machines with 0.8 % Dismozon Plus solution and wipe again with
clean water.
3. Clean all treated areas with 70 % (v/v) ethanol.
4. Change your gloves and continue working.
If the content of a PCR tube spills in the assay run area after the reaction has finished, complete the
following steps:
1. Soak up the spilled liquid with a paper towel.
2. Locally disinfect the area with 70 % (v/v) ethanol.
3. Save and pause all ongoing work or finish analysing the current run if necessary.
4. Clean the whole assay run area following the general protocol.
5. Discard your lab coat when you leave the area and autoclave it before using it again.
23

Protocol 7: Clean-up after a SARS-CoV-2 case among employees
While patients bear the highest risk of carrying a SARS-CoV-2 infection into the mobile lab, they are
restricted to short stays in the patient treatment room. Employees, on the other hand, can move freely in
the whole Mobile Corona Diagnostic Lab and stay for a whole work shift, which increases the chances of
contaminating rooms and equipment. Furthermore, positively tested employees that handled patient
samples could have introduced contamination leading to false-positive test results.
In an employee has been tested positive for SARS-CoV-2 within 24 hours of working in the mobile lab, the
mobile lab has to be temporarily closed for a full decontamination and disinfection according to the
following protocol:
1. Stop all work in the mobile lab, including taking patient swabs. Discard all assays that are currently
being set up or already running as you have to assume they are cross-contaminated.
2. All personal protective equipment worn by the infected employee have to be discarded or
autoclaved.
3. Identify all swabs and extracted RNA obtained in the last 24 hours. Isolate these samples in
dedicated closed containers and label them as potentially contaminated.
4. Discard all opened consumables in every room, such as pipet tips and packages of PCR tubes, as
well as all opened chemicals including open master mixes.
5. Tightly close the trash bags of all table-top waste bins with zip ties or tape and discard them into
the autoclave waste. Close and remove the household trash bag and discard it into the autoclave
waste, too.
6. Close all autoclave waste bags in the lab with zip ties or tape and immediately autoclave them.
7. Disinfect all rooms and work areas of the mobile lab following the general protocol described
above. Pay special attention to also disinfect all surfaces, cupboard fronts, equipment and machines
as well as the inside of all fridges and freezers. Clean the floors last.
8. Discard or autoclave all personal protective equipment of all other lab employees.
9. Clean and disinfect your hands following the hygiene protocol. Put on fresh equipment, including a
new full-body suit or an autoclaved and washed lab coat.
10. Clean all surfaces, all equipment such as pipets and all machines with 70 % (v/v) ethanol. Follow the
work area specific cleaning requirements described above.
11. Restock the mobile lab with sealed consumables such as pipet tips and PCR tubes, open fresh
packages of gloves. Put new trash bags in all table-top waste bins, household waste bins and
autoclave waste bins.
12. You can now re-open the Mobile Corona Diagnostic Lab.
After the mobile lab has been decontaminated and opened again, you will have to re-do all tests from swabs
taken within the last 24 hours or RNA extracted at the same time. Keep these samples in their separate
container and discard them as soon as possible. Contact all patients tested positive in the last 24 hours and
ask them for a new swab sample to confirm the test result.
24

Waste management
Proper management of healthcare waste needs to be based on the waste management
principles and consider integrated and/or holistic management, starting from source
segregation, storage, collection/transport, treatment and final disposal. As per the guidelines
of the CDC (Centers for Disease Control and Prevention), in the United States of America
(USA), healthcare waste generated by COVID-19 patients is considered the same as waste
generated by other patients. Thus, the waste has been treated as regular healthcare waste
and does not require any additional special treatment (Commendatore, 2020).
Figure 4. Overview of solid waste management during COVID-19 pandemic
Segregation
Segregation plays an important role in efficient healthcare waste management . It covers the
separation of different types of waste according to a classification at the point of origin.
Therefore, if segregation of recyclable waste from other non-hazardous waste is carried out
efficiently, it will minimize the waste significantly
To segregate infectious waste, clearly marked containers are used that distinguish the type
and weight of the waste. Infectious waste is generally kept in plastic bags, plastic-lined
cardboard boxes, or other leak-proof containers meeting specific performance standards
following elimination of sharps and fluids. Color-coding is employed to identify different types
of waste easily. In most countries, red or yellow bags are commonly used to contain
infectious waste (Table 9).
26

Table 9: Waste classification and handling instructions
Category Waste Type Bags Type Treatment disposal
Items contaminated
with blood, body fluids
like cotton swab and
containing
components, gown
and routine mask and
gown.
Yellow
coloured, non
chlorinated
plastic bags
first autoclaved and then passed on
to Incineration/plasma pyrolysis,
deep burial. based on the country
regulation,
Chemical waste.
Chemicals used in
production of biological
and used or discarded.
Yellow
coloured, non
chlorinated
plastic bags
Pretreatment before discharge.
based on the norms given in country
regulation
Contaminated waste
(Recyclable)
Wastes generated
from disposable items
such as tubing, bottles,
tubes
Red coloured,
non chlorinated
plastic bags
first autoclaved and then passed on
to Incineration/plasma pyrolysis,
deep burial. based on the country
regulation,
Waste Sharps
including metals.
Objects that may
cause puncture and
cuts.
Puncture proof
leak proof,
tamper proof
containers
Autoclaving, microwaving, followed
by shredding and mutilation
Glassware:Broken or
discarded and
contaminated glass
Puncture proof
leak proof,
tamper proof
containers
Disinfection by detergent such as:
Sodium hypochlorite, autoclaving
and sent for recycling.
Nondisposable
plasticware
Puncture proof
leak proof,
tamper proof
containers
Nondisposable plasticware should
be treated before use to ensure that
it is RNase-free. Plasticware should
be thoroughly rinsed with 0.1 M
NaOH (Sodium Chloride), 1mM
EDTA Ethylenediaminetetraacetic
acid) followed by RNase-free water.
Liquid waste
collected in the
plastic
container
under the sink
in the mobile
lab
chemical inactivation or for only
small volumes autoclavation
27

Autoclave
It must be assumed that all materials and consumables that come into contact with patient
samples are contaminated. Therefore, without exception, all such consumables must be
autoclaved at the end of a working day. For this purpose, the materials such as swab sticks,
reaction vessels, pipette tips and so on are packed in 2 autoclave bags and autoclaved
according to the autoclave manual. then the materials can be disposed of with normal
waste.The autoclave reliably inactivates infectious germs using moist heat. In addition, or for
materials that cannot be autoclaved, incineration is recommended.
Protocol 8: Using the autoclave
Operating Autoclave
1. Close and lock the door.
2. Choose an appropriate cycle (e.g., gravity, liquid, or dry cycle) for the material.
3. Consult the autoclave manual for assistance in choosing a cycle.
4. The manuals for operation of the autoclave should be located near the autoclave.
5. Set appropriate time and temperature if you are using a customized cycle.
6. Start your cycle and fill out the autoclave user log with your contact information.
7. A completed cycle usually takes between 1-1.5 hours, depending on the type of
cycle. Do not attempt to open the door while the autoclave is operating.
8. If problems with your autoclave are perceived, abort cycle and report it to your PI
immediately
Unloading Autoclave
9. Wear heat-insulating gloves, eye protection, lab coat, and closed-toe shoes.
10. Ensure that the cycle has completed and both temperature and pressure have
returned to a safe range.
11. Wearing Personal Protective Equipment (PPE), stand back from the door as a
precaution and carefully open the door no more than 1 inch. This will release
residual steam and allow pressure within liquids and containers to normalize.
12. Allow the autoclaved load to stand for 10 minutes in the chamber. This will allow
steam to clear and trapped air to escape from hot liquids, reducing risk to
operators. Do not agitate containers of superheated liquids or remove caps before
unloading.
13. Wearing heat-insulated gloves remove items from the autoclave and place them in
an area which clearly indicates the items are ‘hot’ until the items cool to room
temperature.
14. Shut the autoclave door.
28

Incineration
Is the high-temperature burning (rapid oxidation) of a waste. It is also known as
controlled–flame combustion or calcination and is a technology that destroys
organic constituents in waste materials
an incinerator is not provided in the mobile lab. Please locate a local incineration station and
follow your local guidelines for disposal.
29

Diagnostic Methods
Patient data management
Please note that the organiser has to provide a patient registration prior to patient sampling,
e.g. by using online apps. The medical technician who takes the sample needs to be able to
assign a patient specific ID to the sample. However, in any subsequent step of the diagnostic
methods, only this ID is used for the samples and the identity of the patient should not be
accessible to the technicians to prevent any personal bias in the evaluation of the samples.
Only at the end when the diagnosis is given to the patient, the result which is determined for
each sample ID the information of patient identity and ID is combined again. The data
management is not part of this handbook. To generate the patient ID following scheme is
recommended: Use the date of swab taking + daily serial number (eg. 19032021-001)
Depending on your local infrastructure and data management concept, you need to integrate
the mobile lab workflow accordingly. If there is no data management, you can maintain an
excel list and track the following information during patients registration (info can be
gathered with a form to fill in before diagnosis)
● full name
● birth date
● means of contact (phone or mail) -> to get result
● address
● date and time of swab taking
● signature
● Patient ID (assigned by staff)
On the back side of the form should be information on the procedure and risks and
information that the patient is willing to give informed consent to the procedure.
After the patient is registered and has an ID, this ID number must be labelled on EVERY
consumable item which is used during the diagnostic procedure - swab sampling, RNA
extraction, RT-LAMP and QPCR and finally also on the Data Readout (Foto and File Export
from QPCR machine). The technicians in the lab are not supposed to have access to the
patient data (not access to the excel sheet) to prevent any personal bias. Only when sticking
to the labelling rigorously the actual result and the patient can be matched and mis
diagnosing be prevented! Thus, make sure you print stickers with the ID or hand label each
vessel in which the patient material will be held in before you start each method. After the
result is obtained and there was no errors, the result and ID are matched with the the patient
information in the excel list. A doctor then has to get in touch with the patient and inform him
/ her about the result as soon as possible.
Patient Sampling
Swabs from patients should be taken in a separate, well-ventilated area referred to as the
treatment room in the following. Only necessary personnel should be present as the direct
close contact with patients bears a high risk of spreading SARS-CoV-2 infections. Only one
30

patient at a time should be present in the treatment room. All further patients have to be
asked to wait outside in the open with sufficient distancing.
The swab samples are taken from the back wall of the throat (oropharyngeal region). This
procedure is non-invasive and not painful, but the procedure will feel unpleasant. Patients
have to register using a form/app and will be assigned an ID number to guarantee a
(double)-blinded sample analysis. For minors, one parent or legal guardian has to fill a
consent form in addition to the registry form. (see patient data management chapter)
Sampling Safety and precautions
All patients are assumed to be SARS-CoV-2 positive until diagnosed otherwise. It is
therefore necessary to follow a strict hygiene protocol and wear personal protective
equipment when taking a swab sample from patients. To keep the risk of contaminations low,
only one patient at a time is allowed to enter the treatment room. Patients may not enter any
other area of the Mobile Lab. The treatment room and all surfaces and equipment have to be
disinfected and cleaned regularly, at least every morning, at noon and in the evening.
Preparing the patient treatment room
The patient treatment room should be cleaned and disinfected as described in the chapter
“Cleaning the Mobile Corona Diagnostic Lab” every morning, at noon or after longer breaks
and every evening. Be sure to disinfect all surfaces touched by the patient after the
procedure including door knobs and arm chairs before the next patient enters. In the evening
or after closing the Mobile Lab treatment room for the day, wipe the floor with 0.8 %
Dismozon Plus solution, wait 15 minutes and wipe the floor again with clean water.
Putting on personal protective equipment
Before you ask a patient to enter the room, put on a complete set of personal equipment as
described in the Protocol 3: Putting on and taking off personal protective equipment. The
patient is only allowed to enter after all personnel present in the room has put on their full
personal protective equipment. For hygiene reasons, all personnel that had direct contact
with the patient should change their gloves before the next patient is treated.
How to take an oropharyngeal swab sample
Oropharyngeal swab samples are taken from the back of a patient's throat using an
approved sterile cotton swab. The procedure will feel unpleasant for the patient although it is
not painful. Keep in mind that touching the back wall of the throat can cause a gag reflex. If
the patient shows strong signs of
nausea such as gulping or
gagging, abort the procedure and
try again after a short break.
The patient has to disinfect his
hands upon entering the Mobile
Lab treatment room and must
wear a mask, which he should
only remove upon request. Ask
the patient not to touch any
31

surfaces or equipment to prevent contaminations.
32

Table 10: Materials list for Patient sampling
Rack for swabs - -
Label printer Brady 139538
Labels for label printer Brady 121016
Table-top waste bin Carl Roth E705.1
Autoclave bags for waste bin Carl Roth E706.1
Fridge set to 4 °C - -
Consumables Included in starter pack
Sterile swab including swab tube Witeg 5475000
sample buffer (PBS or 0.5 % NaCl) - -
Protocol 9: The following procedure should be performed for oropharyngeal swab sampling:
15. Put on your personal protective equipment and prepare the treatment room as described in the
hygiene concept.
16. Prepare an aliquot (3 ml) of sample buffer in a falcon or reaction tube.
17. Ask the patient to enter the treatment room and have him disinfect his hands.
18. Check the patient's identity and prepare a label accordingly. Place the still wrapped swab and the
label close to you for easy reach.
19. Let the patient sit down on a chair and ask them to tilt their head back slightly. It can be helpful to
let them rest their head against a wall.
20. Warn the patient that the procedure will feel unpleasant and that their gag reflex might be
triggered.
21. Stand slightly off-center in front of the patient, do not stand directly in front of them as they might
suddenly sneeze or cough.
22. Ask the patient to take off their mask, open their mouth wide and hold their tounge in a forward
down position. Use a tongue depressor if required.
23. Unpack the sterile swab and carefully move the swab into the mouth cavity without touching the
patient’s lips, teeth, tongue or palate. Support the back of the patient’s neck with your other hand
to prevent them from moving.
24. Take a swab sample from the far back of the throat. Carefully rotate the swab against the
oropharyngeal wall to soak up enough samples.
25. Carefully remove the swab without touching the patient’s lips, teeth, tongue or gums.
26. Put the swab back into its transportation tube, add the prepared aliquot of sample buffer and seal
the tube tightly.
27. Label the tube with the prepared label and wipe the outside with a disinfectant.
28. Ask the patient to put their mask back on. They should now leave the treatment room.
33

29. Check the labeling on the swab again and place the sealed swab in a rack at 4 °C.
Sealed swabs can be stored at 4 °C for a short time before processing them. Transfer swabs into the RNA
working room for RNA extraction and further processing.
Sample Buffer and Storage
Specimens for virus detection should reach the laboratory as soon as possible after
collection. Correct handling of specimens during transportation and in the laboratory is
essential. For transport of specimens for viral detection, use preferentially viral transport
medium (VTM) containing antifungal and antibiotic supplements. If VTM is not available,
other solutions may be used. Such solutions may include phosphate buffered saline (PBS),
0.9% sterile saline (with storage at +4°C up to 7 to 14 days). Per swab a volume of 140 - 280
µl storage buffer is recommended. Transportation of specimens within national borders
should comply with the applicable national regulations. International transport of specimens
that may contain SARS-CoV-2 should follow the United Nations Model Regulations,
Biological Substance, Category B (UN 3373), and any other applicable regulations
depending on the mode of transport.
34

RNA extraction
The ability to purify, analyze, and manipulate RNA is essential for many laboratories working
in the life sciences. There are three major causes of RNA degradation - alkaline pH, divalent
metal ions, and ribonucleases. OXGEN - Viral RNA Extraction Kit provides a streamlined
protocol for avoiding RNA degradation and facilitates isolation of RNA from biological fluids
such as nasopharyngeal and buccal swabs, blood, and others. RNA can be recovered within
30 minutes using a quick and easy micro-spin column purification method and can be
directly used for molecular biology applications such as qPCR, LAMP, reverse transcription
(RT), and targeted RNA sequencing.
The sample is first lysed under highly denaturing conditions to inactivate RNases. Adding
synthetic carrier RNA along as buffering conditions provide an optimum binding of the RNA
to OxGen G-spin® columns. A unique sequence of carrier RNA facilitates RNA binding to
the membrane, and contaminants are efficiently washed away using two different sets of
wash buffers. High-quality RNA is eluted in a special RNase-free buffer, ready for direct use
or safe storage. The purified RNA is free of protein, nucleases and other contaminants and
inhibitors.
Film 3: “RNA extraction”
For further information and protocol details please contact info@oxgen.ge
Table 11: OXGEN Viral RNA Extraction Kit components and storage conditions
Kit Component Label and quantities for 50 rxn Storage
Lysis Buffer Solution A, 30 ml +4 °C or RT
Wash Buffer 1 Solution W 1, 35 ml +4 °C or RT
Wash Buffer 2 Solution W 2, 35 ml +4 °C or RT
Elution Buffer Solution E, 10 ml +4 °C or RT
RNA Carrier Si-RNA, 100 µg/µl -20 °C
OxGen Membrane, with Spin-columns G-spin® columns +4 °C or RT
Table 12: Materials list for Viral RNA Extraction Supplied by User
35

Equipment Included in mobile lab
Benchtop centrifuge (12,000 x g, rotor
compatible with 1.5 ml and 2.0 ml tube)
Hermle Z305.00 V05
Vortexer Ika 0003365000
Pipette 0.5 - 10 µl Neo Biotech NB-12-6001-2
Pipette 10 - 100 µl Neo Biotech NB-12-6001-5
Freezer set to -20 °C - -
1.5 ml and 2.0 ml tubes with cap, low binding,
certified ribonuclease-free
Eppendorf 0030120086
Benchtop cooler or ice in ice box (for short-term
storage of RNA)
Eppendorf
Pipette tips with filter, 0.5 - 10 µl Eppendorf 0030078519
Pipette tips with filter, 2 - 200 µl Eppendorf 0030078551
Paper tissue roll Stier 91913986
ethanol, diluted to 96-100 % (v/v) Carl Roth T913.3
Protocol 10: RNA extraction protocol from swabs or buccal swabs
The OXGENViral RNA Extraction Kit contains five buffer solutions as well as spin-columns and eppendorf
reaction tubes (see table 11).
Before you start
1. Put on fresh gloves and protective clothes as described in the Protocol: Putting on and taking off
personal protective equipment.
2. Clean the workspace and all equipment as described in the chapter Cleaning the Mobile Corona
Diagnostic Lab. You have to keep your workspace free of both RNAses and contaminating RNA from
now on.
3. Add 260 μl Solution E to 210 μg lyophilized siRNA carrier to obtain a solution of 0.84 μg/μl. Aliquot
dissolved RNA carriers and store at -20 °C. Do not freeze-thaw more than five times.
4. Buffer W1 and W2 are supplied as a concentrate. Before using for the first time, add the
appropriate amount of ethanol (96–100%).
Wash buffer preparation
Solution Number of Preps/ 50 Number of Preps/ 250
Wash 1 12.5 ml + 22.5ml ethanol (96–100%) 62.5ml + 112.5ml, ethanol (96–100%)
Wash 2 3.5ml + 31.5ml ethanol (96–100%) 17.5 + 157.5 ethanol (96–100%)
RNA extraction protocol
36

5. Calculate the amount of Solution A (560 µl/reaction) and RNA Carrier (5 µl/reaction) required for
the run, transfer the required amounts into a reaction tube and vortex well to mix. For each
sample, transfer 565 µl of the mixed solution into labelled microcentrifuge tubes.
6. Add 140 µl sample to the respective tube. Mix by pulse-vortexing for 15 sec. If the sample volume is
larger, the amount of Solution A and RNA Carrier have to be increased proportionally. If the sample
volume is less than 140 µl, adjust with PBS.
Incubate for 10 min at room temperature.
7. Briefly centrifuge the tube to remove drops from inside the lid.
8. Add 560 µl of ethanol (96–100%) to the sample and mix by vortexing for 15 sec. Briefly centrifuge
the tube to remove drops from inside the lid.
9. Transfer 650 µl of lysate onto a G-spin® column placed in a 2.0 ml collection tube and centrifuge at
8,000 rpm for 1 min.
10. Transfer the rest of lysate onto a G-spin® column and centrifuge at 8,000 rpm for 1 min. Transfer
the spin-column into a new 2 ml collection tube.
11. Add 600 µl Solution W1 to the spin-column and centrifuge at 8,000 rpm for 1 min. Discard the flow
through and transfer the spin-column into a new 2 ml collection tube.
12. Add 600 µl Solution W2 to the spin-column and centrifuge at 13 000 rpm for 3 min. Transfer the
column into a sterile, RNAse-free 1.5 ml eppendorf reaction tube.
13. Add 50 µl of Solution E on the column and ensure that the entire surface of the column is hydrated.
Incubate for 3 min at room temperature.
14. To elute RNA from the spin-column, centrifuge at 8,000 rpm for 1 min. Viral RNA is stable for up to
one year when stored at -80 °C.
RT-qPCR
The reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique is
used to amplify viral RNA for fluorescent read-out. This method is considered the
gold-standard for diagnosing a SARS-CoV-2 infection and is recommended by the World
Health Organization. The RT-qPCR consists of several steps, which have to be performed at
different temperatures. First, a reverse transcriptase transcribes a small fraction of the
single-stranded SARS-CoV-2 RNA into double-stranded cDNA. This template is then
amplified within 30-40 repetitive reaction cycles to generate a large amount of DNA copies.
The accumulation of DNA copies can be followed with a fluorescent signal and is used to
compute the amount of initial viral RNA in the sample. This feature enables a quantitative
estimate how far the SARS-CoV-2 infection has progressed.
37

Figure 5: RT-QPCR schematic
While RT-qPCR is a highly sensitive method and enables a quantification of the
SARS-CoV-2 infection state, it requires trained personnel and well-equipped laboratories
with specialized equipment. The method is therefore only of limited use for POC diagnostics.
Film 6: “RT-qPCR setup”
It is recommended to run the Chaibio QPCR device of Mobile Corona Diagnostic Lab with
the RT-QPCR kit AllplexTM
SARS-CoV-2 Assay RT-qPCR kit for Covid-19 diagnostics of the
manufacturer “Seegene”. This kit enables multiplexed detection of two different targets within
the SARS-CoV-2 RNA.
RT-qPCR kit
The AllplexTM
SARS-CoV-2 Assay RT-qPCR kit (Seegene) [ref] enables a sensitive detection
of SARS-CoV-2 RNA after highly specific nucleic acid amplification and accumulation. The
assay can target subregions of the E-gene, RdRP-gene and N-gene in parallel and has an
internal positive control (MS2 phage gene) included.
The AllplexTM
SARS-CoV-2 Assay RT-qPCR kit (Seegene) contains two enzymes in a
suitable reaction buffer to produce a vast amount of DNA copies from different small regions
of the viral genetic information. The kit also includes reporter probes marked with different
fluorescent dyes (fluorescein, CAL Red 610, Quasar 670, HEX), which lead to an increasing
fluorescent signal with progressing DNA accumulation. The HEX reporter probe only shows
an increase in signal when the included positive control is accumulated. It serves as an
internal control to verify that the RT-qPCR run is valid. The fluorescein, Cal Red 610 and
Quasar 670 reporter probes show an increase in signal when the respective SARS-CoV-2
genes are amplified and accumulated and serve as read-outs for SARS-CoV-2 infections.
Specifications and limitations
The AllplexTM
SARS-CoV-2 Assay RT-qPCR kit (Seegene) enables a reliable detection of
SARS-CoV-2 infection for individuals with an RNA concentration of down to 4.2 copies per
µl. The kit should be used with extracted RNA purified from nasopharyngeal swabs.
Results reflect the current infection state of the tested person. Reliable results can only be
obtained if proper procedures for swab collection as well as transport, storage and
38

processing of samples and kit are followed. Individuals with invalid, questionable or
non-interpretable test results should repeat the RT-qPCR assay.
The Mobile Corona Diagnostic Lab is delivered with a dual-channel RT-qPCR instrument
and can thus only be used for the parallel detection of E-gene and internal control. Detection
of all included targets is only possible in combination with other RT-qPCR instruments
allowing parallel detection of all four included fluorophores.
Table 13: Materials list RT-qPCR on extracted RNA samples
Rack for PCR tubes Carl Roth HX25.1
Metal cooling block for PCR tubes Eppendorf 3881000015
RT-qPCR machine Chai Biotechnologies E013201
laptop / tablet Samsung Galaxy Tab S7 WiFi
PCR tubes 0.2 ml, with attached lids Eppendorf 0030124359
AllplexTM
SARS-CoV-2 RT-qPCR kit Seegene RV10248X
nuclease-free water ThermoFisher AM9935
SARS-CoV-2 positive control Twist Bioscience 102024
patient samples - -
Protocol 11: Setting up an RT-qPCR run with extracted RNA
The AllplexTM
SARS-CoV-2 Assay RT-qPCR kit (Seegene) contains five different components sufficient to
prepare mastermixes for 100 reactions as well as nuclease-free water.
Before you start
39

now on.
3. Place a metal cooling rack at -20 °C in advance to pre-chill it.
4. Prepare one sterile PCR tube with closed lids for each sample and label it according. Ensure you
have additional PCR tubes for positive and negative controls.
5. Prepare a sterile 1.5 ml reaction tube for the mastermix and label it accordingly.
6. Thaw all reagents of the kit on ice, vortex to mix them and spin down briefly.
7. Dilute the included positive control 1:10 with nuclease-free water and freeze as small aliquots.
Preparing the mastermix and setting up the reactions
8. Place the prepared sterile PCR tubes in the pre-cooled metal cooling rack.
9. Calculate the number of reactions n required for this run. Consider the number of patient samples
as well as positive and negative controls and add 2 additional reactions to account for any pipetting
inaccuracies.
→ n = # samples + 1 positive control + 1 negative control + 2 additional reactions
10. Pipet the required amount of all kit components for n reactions in the following order:
a. SARS2 MOM: n * 5.0 µl primer mix
b. EM8 n * 5.0 µl enzymes and buffer
c. nuclease-free water n * 4.8 µl
d. RP-V IC: n * 0.2 µl internal control
11. Briefly vortex the mastermix and spin the tube down.
12. Add 15 µL of the mastermix to each prepared PCR tube. Close the tubes to avoid carryover
contaminations.
13. Add 5 µL nuclease-free water into the PCR tube assigned as negative control. Mix by pipetting up
and down three times and tightly close the lid.
14. Add 5 µL sample to the correct PCR tubes, mix by pipetting up and down three times and tightly
close the lid. Ensure you add the samples to the correct tubes.
15. Add 5 µL of the diluted positive control to the correct PCR tube assigned as positive control. Pipet
up and down three times and tightly close the lid.
Running and analyzing an RT-qPCR run
The AllplexTM
SARS-CoV-2 Assay RT-qPCR kit (Seegene) is a fluorescent assay. The
Chaibio open qPCR software automatically analyzes all fluorescent curves and calculates
cycle threshold (Ct) values where appropriate. If SARS-CoV-2 specific nucleic acids are
present in a sample, the fluorescent signal in the FAM channel for this reaction will increase
exponentially due to the exponential DNA amplification.
Film 7: “qPCR run + analysis”
40

The Chaibio open qPCR software automatically determines a value for the constant
fluorescence background noise, which does not change significantly over the entire cycling
time and is called the threshold value. If the fluorescent signal in a reaction increases
exponentially, it will turn bigger than the threshold value and the sample is considered
SARS-CoV-2 positive. In this case, the software automatically calculates the Ct value of the
reaction. The Ct value matches the cycle at which the fluorescent signal becomes bigger
than the background threshold value. The RT-qPCR runs 45 cycles in total, if the fluorescent
signal of a reaction does not increase above the threshold value after 40 cycles the sample
is considered to be SARS-CoV-2 negative.
Protocol 12: Running the RT-qPCR assay
1. Open the ChaiBio Open qPCR Software and select "New experiment".
2. Select stage 1 (holding) and program the following parameters:
a. step 1: 50 °C, 20 min
b. step 2: 95 °C, 15 min
3. Select stage 2 (cycling) and program the following parameters:
a. step 1: 94 °C, 15 sec
b. step 2: 58 °C, 30 sec; detect fluorescence
c. Cycling: 45x
4. Select stage 3 (holding) and program the following parameter:
a. step 1: 4 ° C, 277 h
5. Ensure that both FAM and HEX channels are selected.
6. Place the PCR tubes in the Open qPCR Dual Channel instrument (Chai Biotechnologies Inc.) and
check again that all lids are closed tightly.
7. Start the run (top right corner). A new window is shown where the run progress can be followed.
8. Label all tubes with the patient ID in the software.
Documenting the results:
● export data, save
The AllplexTM
SARS-CoV-2 Assay RT-qPCR kit (Seegene) includes an internal positive
control, which yields a fluorescent signal in the HEX channel. The internal positive control is
analysed for each reaction in parallel to the SARS-CoV-2 specific fluorescent signal. Thus,
each reaction can be assigned two individual Ct values: one for the internal control and one
specific for SARS-CoV-2.
41

Figure 6: RT-QPCR result interpretation
It has to be noted that the fluorescent curves can be displayed in a logarithmic or linear
fashion. This does not affect the threshold value or the Ct values calculated by the software.
The assay can only be interpreted reliably if the following criteria are all met:
● A constant fluorescent background value (threshold value) can be defined, AND
● the negative control reactions were assigned a Ct value > 40, AND
● the internal positive control yields a Ct value < 40.
If all three of the above criteria are met, the run is considered to be valid and the reactions
can be analyzed. This validity check has to be performed for each run individually,
independent of the mastermix lot. If the run is valid, all samples that have been automatically
assigned a Ct value in the FAM fluorescent channel are considered to be SARS-CoV-2
positive. Samples where the FAM fluorescent signal did not increase exponentially above the
threshold value and have thus not been assigned a Ct value are considered to be
SARS-CoV-2 negative.
A run is considered invalid if one of the following criteria are met:
● No stable threshold value could be defined, AND/OR
● the negative control reactions were not assigned a Ct value > 40, AND/OR
● the internal positive control did not yield a Ct value < 40 for all samples.
When a run is considered invalid, the included reactions cannot be judged safely. To obtain a
reliable result, all samples have to be tested again in a new run. Possible reasons for the
failure can be found below in the troubleshooting section.
42

RT-LAMP
Reverse transcription coupled loop-mediated isothermal amplification (RT-LAMP) is an
alternative method for nucleic acid amplification and can be used as a diagnostic test to
detect SARS-CoV-2. The method is used to transcribe single-stranded SARS-CoV-2 RNA
into double-stranded cDNA, which is then amplified to accumulate short pieces of
SARS-CoV-2 specific DNA.
Figure 7: RT-LAMP schematic
RT-LAMP is a particularly suitable method for on-site testing as it is a one-pot reaction that
can be conducted at constant temperature within 30-45 minutes and thus requires only
minimal equipment. Moreover, six different DNA oligonucleotides (primers) are required for
amplification, making the overall process highly specific for SARS-CoV-2 RNA and
minimizing the risk of detecting contaminants. The accumulated DNA pieces can be read out
by different methods including fluorescence, turbidity and colorimetry, thus making RT-LAMP
a flexible method adaptable to the respective local premise.
Film 4: “RT-LAMP assay setup”
The DisCoV2er RT-LAMP mastermix, which was developed for the Mobile Corona
Diagnostic Lab, combines all required enzymes for the RT-LAMP reaction with a suitable
buffer and an oligonucleotide set specific for a certain region of the SARS-CoV-2 RNA. The
supplied dye mixture leads to a color change which is visible by naked eye and thus allows a
simple and fast colorimetric read-out. The color change is based on changes of the buffer
ion concentration upon accumulation of SARS-CoV-2 DNA.
RT-LAMP kit
The DisCoV2er RT-LAMP kit allows fast and sensitive detection of SARS-CoV-2 RNA after
nucleic acid amplification and accumulation. The assay targets a subregion of the envelope
gene with high specificity, a crossreaction with other coronaviruses is not known.
The DisCoV2er mastermix contains two enzymes in a suitable reaction buffer, which
produce a vast amount of copies from a small region of the viral genomic information. The
enzymatic activities in the presence of viral RNA deplete the reaction of magnesium ions
(Mg2+
), which are included in the reaction buffer in a tightly controlled amount. The dye
43

mixture also included in the assay mastermix is sensitive for magnesium ion changes and
leads to a visual color change from red to green if viral RNA was present in the sample. This
allows an easy visual analysis of samples with a clear and easy to interpret readout.
Specifications and limitations
The DisCoV2er RT-LAMP kit enables a reliable detection of SARS-CoV-2 infection for
individuals in the infectious stage with a viral load of at least 1000 genome equivalents per
ml or an RNA concentration of 5 copies per µl. This corresponds to a RT-qPCR Ct value of
36. The DisCoV2er assay should be used with extracted RNA purified from nasopharyngeal
swabs. The assay is qualitative and not suitable for quantification of the viral load.
Results reflect the current infection state of the tested person and are not valid for a
prolonged time. Reliable results can only be obtained if proper procedures for swab
collection as well as transport, storage and processing of samples and kit are followed. A
negative result does not rule out a SARS-CoV-2 infection at early or late stages with low viral
loads. It is therefore recommended to repeat the RT-LAMP test after 3-5 days. Individuals
with invalid, questionable or non-interpretable test results should repeat the RT-LAMP assay
or be retested using a RT-qPCR kit.
Table 14: Materials list for RT-LAMP with extracted RNA
Rack for PCR tubes Carl Roth HX25.1
Metal cooling block for PCR tubes Eppendorf 3881000015
Dry Bath Incubator Neo Biotech NB-12-0008A
Block 96x 0.2 ml for dry bath incubator Neo Biotech NB-12-0008-01
camera / photo setup - -
PCR tubes 0.2 ml, with attached lids Eppendorf 0030124359
RT-LAMP master mix DisCoV2er master mix -
44

SARS-CoV-2 positive control Twist Bioscience 102024
Protocol 13: Setting up RT-LAMP reactions with extracted RNA
The DisCoV2er RT-LAMP kit is delivered as a ready-to-use mastermix containing all required components.
After addition of a sample to the mastermix, the reaction can directly be incubated and results can be
obtained after 45 minutes incubation time.
Before you start:
now on.
3. Place a metal cooling rack at -20 °C in advance to pre-chill it.
4. Prepare one sterile PCR tube with closed lids for each sample and label it accordingly. Ensure you
have additional PCR tubes for positive and negative controls.
5. Obtain a frozen mastermix as well as an aliquot of RNA positive control directly before you start
setting up the reactions.
Setting up the test reactions of RT-LAMP
6. Place the prepared sterile PCR tubes in the pre-cooled metal cooling rack.
7. Carefully thaw the mastermix until it is completely liquid. Vortex well for 10 seconds.
a. → Inspect the mastermix: It should have a deep red color and no precipitates or flakes
should be visible.
8. Add 17 µL of the prepared mastermix in each PCR tube. Close the tubes to avoid carryover
contaminations.
9. Add 8 µL nuclease-free water into the two PCR tubes assigned as negative controls. Mix by
pipetting up and down three times and tightly close the lid.
a. → Ensure you include two negative controls in each individual run and check that the
reaction is still red in color.
10. Add 8 µL sample to the correct PCR tubes, mix by pipetting up and down three times and tightly
close the lid. Take care to add the samples to the correct tubes.
a. → Check that the reaction is still red in color after sample addition.
11. Add 8 µL of synthetic RNA (60 cp/µL stock concentration) to the correct PCR tubes assigned as
positive controls. Pipet up and down three times and tightly close the lid.
a. → Ensure you include two positive controls in each individual run and check that the
reaction is still red in color.
Running and analyzing an RT-LAMP assay
45

Film 5: “RT-LAMP run and analysis”
Protocol 14: Running and documenting the RT-LAMP assay
1. Place all PCR tubes in a heat block. Double-check that all lids are tightly closed.
2. Set the program to incubate the samples at 65 °C for 45 minutes and check that the instrument lid
heating is turned on.
3. Start the incubation.
Documenting the results of RT-LAMP
4. After the incubation time, immediately cool the samples down to 4 °C either in the heat block or in
the metal cooling rack.
5. → When you take out the samples, ensure the PCR tube lids do not pop open. Do not open the PCR
tubes anymore.
6. Visually check the samples for their color change and note down your findings:
7. Check whether both negative controls remained red.
8. Check whether both positive controls turned green.
9. Check the color of each individual sample by comparing it to the positive and negative controls and
label the sample color as green, red, or indecisive.
10. Place the samples on a white background and take a picture. Ensure that the colors are depicted
realistically. Label and save the picture according to the data management guidelines (chapter x).
11. Discard the PCR tubes safely. Ensure that the lid has not been opened again after the reaction has
been incubated.
The DisCoV2er RT-LAMP assay is a colorimetric assay that can be judged by naked eye
based on its color change. The assay can only be interpreted reliably if the following three
criteria are all met:
● All reactions were red prior to incubation, AND
● the negative control reactions remained red after incubation, AND
● the positive control reactions turned green after incubation.
If all three of the above criteria are met, the respective run is considered valid and the
reactions included in the respective run can be analyzed. This validity check has to be
performed for each run independent of the mastermix lot.
When a run is considered valid, the colors of all individual reactions are judged as red,
green, or indecisive compared to the positive and negative controls included in the run. A
reaction is inconclusive if its color is neither clear red nor clear green. The color of
inconclusive reactions is often described as brown, (dirty) orange, or mud-colored.
46

Figure 8: Schematic for interpretation of RT-LAMP results
A run is considered invalid if one or more of the following criteria are met:
● The positive control remained red after incubation, AND/OR
● the negative control turned green after incubation.
When a run is considered invalid, the included reactions cannot be judged safely. To obtain a
reliable result, all samples have to be tested again in a new run. Possible reasons for the
failure can be found below in the troubleshooting section.
Troubleshooting
While the DisCoV2er RT-LAMP kit was found to be a robust and reliable test, mistakes and
errors in handling the mastermix or samples, or non-sterile working environments can lead to
invalid results. Below are the most common problems listed which can occur as well as
possible solutions.
The positive control remained red. A red positive control suggests that either no RNA was
added or the RT-LAMP reaction was inhibited.
● The synthetic RNA was degraded: Aliquot the positive control RNA to avoid repeated
freeze-thaw cycles. Store RNA at -80 °C to prevent degradation. Use only
RNAse-free equipment and material when handling RNA.
● The reaction was inhibited: Check that the added RNA did not contain inhibitors or
interfering compounds such as EDTA or magnesium. Samples should not contain
SDS or high concentrations of guanidine.
● Faulty mastermix composition: Vortex the RT-LAMP mastermix well after thawing to
ensure a proper mixing of its ingredients. Check that no flakes or precipitates remain.
● The set-up was incorrect: Check that the instrument settings are correct, most
importantly that the temperature was not above 65 °C at any time.
The negative control turned green. This observation suggests that a contamination has
occured.
● The added nuclease-free water is contaminated: Discard the used aliquot and open a
fresh, unused one.
● The provided RT-LAMP mastermix is contaminated: Try to use a new lot of unused
mastermix. Aliquot to prevent repeated freeze-thaw cycles of the mastermix.
● A carry-over contamination from another reaction has occurred: Always pipet the
positive control first before handling any RNA, close the tubes tightly and do not open
again.
● Reactions were incubated for too long: Ensure that the reactions are not incubated at
65 °C for more than 45 minutes. The reactions have to be cooled to 4 °C directly after
47

incubation to stop the enzymatic reaction. Make sure to analyze the reactions directly
after cool-down.
● An environmental contamination has occured: Clean the workbench and all surfaces
as well as all equipment prior to setting up the reactions. Clean the heatblock and its
lid prior to incubation. Use a bleach-containing detergent and a RNAse-removing
agent for all cleanings and wipe again with 80 % EtOH before starting to work.
The color change of many samples is indecisive.
● The reaction is delayed: Check whether the samples contain interfering compounds
such as EDTA, magnesium or incompatible buffers. Dilute the samples with
nuclease-free water to dilute possible inhibitors. Samples should not contain SDS or
high concentrations of guanidine.
● The RNA concentration is low: If possible check the RNA concentration and
concentrate the sample. Samples below the limit of detection can not be detected
reliably.
● The mastermix has been compromised: Confirm that the mastermix has been
handled and stored properly and that it has not been thawed and frozen repeatedly.
Use a fresh aliquot of unused mastermix to repeat the run.
Some/all reactions turned green upon addition of the sample.
● The samples contain high concentrations of magnesium: Ensure the samples are
delivered in nuclease-free water, PBS, 0.9 % NaCl or any other compatible buffer.
● The mastermix is faulty: Confirm that the mastermix has been prepared properly and
includes the correct concentration of all additives. Confirm that the mastermix has
been handled and stored properly. Use a fresh aliquot of unused mastermix to repeat
the run.
48

Abbreviations
COVID-19 coronavirus disease 2019
RT-LAMP Reverse transcription loop-mediated isothermal amplification
RT-qPCR Reverse transcription quantitative polymerase chain reaction
SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
UVGI Ultraviolet germicidal irradiation
WHO World Health Organization
POC Point-of-Care
References
https://www.nature.com/articles/s41586-020-2196-x
https://pubmed.ncbi.nlm.nih.gov/31986264/
https://www.sciencedirect.com/science/article/pii/S1743919120301977
https://www.nature.com/articles/s41564-020-0695-z
https://www.sciencedirect.com/science/article/abs/pii/S0140673620301835
https://www.sciencedirect.com/science/article/pii/S1876034120304329#bibl0005
Appendix
Educational Videos
Film 1: “Setting up the mobile Lab”
Describes how the lab is set up, gives a general overview regarding functions and
equipment of the mobile hab. Shows the room concept and unidirectional workflow
Film 2: “Hygiene concept”
Describes how personal protective equipment is used and how the lab has to be cleaned in
order to prevent cross contaminations
Film 3: “RNA extraction”
describes how the patient samples are further processed after sample collection. Shows how
RNA is extracted with the Oxgen Kit.
Film 4: “RT-LAMP assay setup”
Shows handling and pipetting steps needed for RT-LAMP assay
Film 5: “RT-LAMP run + analysis”
Shows how to carry out RT-LAMP, control the heat block and how to analyze results of the
assay.
49

Film 6: “RT-qPCR setup”
Shows handling and pipetting steps needed for RT-qPCR assay
Film 7: “qPCR run + analysis”
Shows how to carry out qPCR, control and programming the thermocycler and how to
analyze results of the assay.
Starter Pack Inventory
Table 15: Starter Pack Materials list for the mobile lab
included in Starter Pack Producer Model Storage
Dismozon Plus Bode 981257
room
temperature
Labels for label printer Brady 121016
room
temperature
autoclave bag for waste
bin Carl Roth ATY4.1
room
temperature
Autoclave bags for
waste bin Carl Roth E706.1
room
temperature
room
temperature
Full-body protective
suit DuPont Model Tyvek
room
temperature
PCR tubes 0.2 ml, with
attached lids Eppendorf 30124359
room
temperature
Pipette tips with filter,
0.5 - 10 µl Eppendorf 30078519
room
temperature
Pipette tips with filter,
2 - 200 µl Eppendorf 30078551
room
temperature
room
temperature
clean water Oxgen on request
room
temperature
ethanol, diluted to 70
% (v/v) Oxgen on request
store in chemical
cabinet
ethanol, diluted to
96-100 % (v/v) Oxgen on request
store in chemical
cabinet
RNase Erase Oxgen on request store in chemical
50

cabinet
RT-LAMP DisCoV2er
master mix Oxgen on request
store at -20 °C,
avoid repeated
freeze-thaw
cycles
sample buffer (PBS or
0.5 % NaCl) Oxgen on request store at 4 °C
SARS-CoV-2 positive
control Oxgen on request store at -20 °C
Skin cleaning agent Oxgen on request
room
temperature
Skin disinfectant agent Oxgen on request
room
temperature
Sterile swab including
swab tube Oxgen on request
room
temperature
1.5 ml tubes with cap,
low binding, certified
ribonuclease-free Oxgen on request
room
temperature
2.0 ml tubes with cap,
low binding, certified
ribonuclease-free Oxgen on request
room
temperature
RNA-Extraction kit Oxgen on request
store at -20 °C
AllplexTM SARS-CoV-2
RT-qPCR kit Seegene RV10248X
store at -20 °C
duct tape Spada
5201874545201
2
room
temperature
room
temperature
room
temperature
Chemical Safety instructions
In accordance with Regulation (EC) No. 1907/2006 of the European Parliament and the Council (REACH) and Commission
Regulation (EU) No. 830/201
The following components of the Oxgen Viral Rna Extraction kit contain hazardous contents.
A MSDS document detailing all hazardous and handling instructions can be found at
www.oxgen.ge
51

Book 2 Mobile Corona Diagnostic Lab Manual.pdf

Book 2 Mobile Corona Diagnostic Lab Manual.pdf

Recommended

Recommended

More Related Content

Similar to Book 2 Mobile Corona Diagnostic Lab Manual.pdf

Similar to Book 2 Mobile Corona Diagnostic Lab Manual.pdf (20)

More from ssuser432659

More from ssuser432659 (20)

Recently uploaded

Recently uploaded (20)

Book 2 Mobile Corona Diagnostic Lab Manual.pdf