Participants of the workshop learn the necessary background information and techniques to diagnose Sars-CoV-2 using the mobile diagnostic laboratory. The laboratory 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. Experts accompany the learning process with written teaching materials, video training, virtual live coaching, and short exams to verify the learned content.

1. MOBILE CORONA DIAGNOSTIC LAB
THE PRINCIPLES OF
PCR, RT-QPCR AND RT-LAMP

2. BLOCK 3: RT-LAMP

3. LAMP: LOOP-MEDIATED ISOTHERMAL AMPLIFICATION
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• Alternative technique for the amplification of DNA (or RNA when couples with reverse transcription)
• First reported in 2000 by Tsugunori Notomi
• Applications:
o Screening assays in the field
o Diagnosis of infectious diseases, e.g. ZikaVirus, tuberculosis, malaria, sleeping sickness, …
• Benefits compared to conventional PCR:
o Isothermal → no temperature cycling required, no need for a thermocycler
o Low-cost technique, compatible with complex samples such as blood
• Limitations compared to conventional PCR:
o Not suitable for cloning applications
o Complicated primer design, constrains in choosing the target site
o Multiplexing is very difficult

4. HOW DOES IT WORK?
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management

5. PRIMER DESIGN
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• LAMP requires a set of 4 primers to work (2 forward, 2 backward)
• Including an additional set of loop primers further enhances the reaction
• Define 3 regions on each strand + 1 additional for the loop primer

6. THE LAMP REACTION MECHANISM 1 – LOOP FORMATION
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• Strand invasion by forward inner primer and synthesis of complementary strand
➢ Introduction of new region F3 at 5’ end
• Strand invasion by forward external primer leads to displacement of new strand
➢ First loop forms at 5’ end

7. THE LAMP REACTION MECHANISM 2 – DUMBBELL STRUCTURE
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• Reverse inner primer binds to newly synthesized strand → synthesis of complementary strand
➢ Introduction of new region B3 at 5’ end
• Strand invasion by reverse external primer leads to displacement of new strand
➢ Second loop forms at 5’ end

8. THE LAMP REACTION MECHANISM 3 – LAMP CYCLING
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• Dumbbell structure contains multiple sites for DNA synthesis initiation:
o 3’ end of loops
o Inner primers
o Loop primers

9. DETECTION OF LAMP PRODUCTS
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• Fluorescence (intercalating dye)
• Agarose gel: ladder pattern
• Turbidity: precipitation of magnesium pyrophosphate
• pH-sensitive dyes: e.g. phenol red (NEB)
• Metal-sensitive indicators: e.g. hydroxynaphtol blue, calcein, malachite green

10. RT-LAMP IN THE MOBILE CORONA DIAGNOSTIC LAB
July 8th, 2021
MOBILE CORONA DIAGNOSTIC LAB | Diagnostic Methods and Data Management
• RT-LAMP master mix contains two enzymes, buffer, dNTPs, MgSO4, betaine, a LAMP primer set and two dyes
• SARS-CoV-2 targets: E-gene or N-gene (optional)
• RT-LAMP has to be incubated at 65 °C for ~ 40 min
• Read-out: colorimetric, color change from red (negative) to green (positive)