This document provides information on laboratory testing for Covid-19 using RT-PCR. It describes how RT-PCR works to detect the presence of the SARS-CoV-2 virus by converting its RNA to DNA and amplifying any viral DNA present in samples through repeated cycles. Nasal and throat swabs are common sample types, and results are usually available within 4-8 hours. A lower Ct value indicates a higher viral load was present in the sample. Safety precautions like PPE and proper sample handling are important when conducting these tests.

1. Laboratory Test –
Covid 19
M.C.E.LEEMA MSC., M.PHIL.
MICROBIOLOGY

2. What is covid 19
It is a pandemic(all over the world)disease caused by Coronavirus
RNA virus
It is also called novel coronavirus because it is a newly discovered species
Differ from the old species
Symptoms – fever, dry cough, tiredness (common)
Aches, sorethroat, diarrhoea, conjunctivitis, loss of taste or smell, discoloration of fingures (less
common)

3. SAFETY PRECAUTIONS
 Always use personal protective equipment (PPE) e.g. Laboratory
apron/gown, face mask, gloves and goggles.
 Wipe gloves thoroughly with a disinfectant (e.g., surgical spirit
before and after taking the sample.

4. Specimens or Samples
TWO TYPES
1.Upper respiratory specimens:
 nasopharyngeal and oropharyngeal swab
2.Lower respiratory specimens:
 sputum (if produced) and/or endotracheal aspirate or bronchoalveolar lavage in patients
with more severe respiratory disease. (Note high risk of aerosolization)
 In case of patients who are deceased, consider autopsy material including lung tissue.
 In surviving patients, paired serum (acute and convalescent) can be useful to
retrospectively define cases as serological assays become available

5. Sample containers

6. Nasal swab collection
 Take a fresh sterile swab
 Gently tilt the patient’s head Backwards and steady the chin
 Insert the swab into the nostril parallel (1-2 cm) to the palate
until the resistance is met at turbinate
 Hold the swab in that position for few seconds and then
withdraw slowly in a firmly rotating motion (5 times clockwise
and 5 times anticlockwise).
 The nasal swab is placed in the same VTM tube containing
throat swab.
 The applicator stick is broken off as done for throat swab and
the tube is screw capped tightly.

7. Throat/ Oro-pharyngeal swab collection
 Gently tilt the patient’s head back
 Steady the chin
 Ask the patient to open his/her mouth
 Use a disposable tongue depressor to hold the tongue well
 Insert a sterile swab
 Swab both the tonsils and the posterior pharynx vigorously with a
rotating motion, till the patient starts to gag
 Remove the swab without touching the tongue.
 The swab is then placed in the labelled tube containing VTM
 The applicator stick is broken off at the indicated mark (if
provided)or at below the level of the tube opening
 Close and tightly screw cap the tube.

8. Transporation
 Specimens for virus detection should reach the laboratory
as soon as possible after collection.
 Correct handling of specimens during transportation is
essential.
 Specimens that can be delivered promptly to the
laboratory can be stored and shipped at 2-8°C.
 When there is likely to be a delay in specimens reaching
the laboratory, the use of viral transport medium is
strongly recommended.
 Specimens may be frozen to - 20°C or ideally -70°C and
shipped on dry ice if further delays are expected
 Avoid repeated freezing and thawing of specimens.

9. Current testing modalities for
laboratory diagnosis of COVID-19
Real Time RTPCR testing is the recommended testing for COVID-19
diagnosis.
What is rRT-PCR?
RT-PCR test is a real-time reverse transcription polymerase chain
reaction (rRT-PCR)
It is qualitative detection of nucleic acid from SARS-CoV-2 in upper and
lower respiratory specimens.
 It is loaded with fluorescent dyes as markers.
 It detects the virus even if the viral load is less.
 The major advantage of Real-Time RT-PCR is that it gives rapid results.

10. What is the procedure for RT-PCR Test for the
COVID-19 virus?
Step 1:- The COVID-19 virus generally gathers in the throat or inside the nose of a
person.
Step 2:- The cells and nucleus are lysed by treating them with different chemical
solutions. Finally, the sample only contains DNA and RNA of the virus.
Step 3:- The RNA is then converted to DNA using a specific enzyme reverse
transcriptase.
Short fragments of DNA(complementary DNA) are also added to create a mixture.

11. Continues…
 Step 4:- If there is a virus in the sample, then the short fragments of the DNA
accord to the target divisions of the viral DNA.
 Step 5:- The mixture is then put into an RT–PCR machine.
 The RT-PCR machine ignite specific chemical reactions.
 Through this process, the target sections of viral DNA get their new identical
copies.
 Step 6:- Repeated cycles carried out with the sample. At the end of each
cycle, the number of copies of the viral DNA gets doubled.
 As a result, about 35 billion new copies of the viral DNA sections from each
strand of the virus are generated by the end of the process.
 The RT-PCR testing time is only 5-10 minutes, but the entire procedure takes
upto 4 to 8 hours.

12. How is the result concluded?
 Once the new copies of the viral DNA are formed, the marker labels then attach to
the DNA strands.
 The RT-PCR machine is connected to a computer that records the amount of
fluorescent dye released after each step.
 At a certain point, when the amount of fluorescence crosses a particular limit, this
confirms the presence of the virus.
 In the fewer the cycles to confirm the virus, the more severe is the condition.
 RT-PCR test report time can take 4 to 8 hours.

13. continues
 Initial Screening RT PCR involves detection of ‘E’ gene (coding for SARSCoV-2
viral envelope)
 Confirmation of screening PCR involves detection of
one of the following two gene targets:
RdRp gene (coding for SARS-CoV-2 RNA dependent RNA
Polymerase)
ORF gene (coding for SARS-CoV-2 Open Reading Frame)
What is CT value?
The Cycle Threshold value is basically the number of cycles needed for the virus to be
detected from the person's sample.

15. Continues…
Results and interpretation:
 A Ct value 29 or below- high viral load
 A Ct value 30-37 - moderate viral load
 A Ct value 38-40 - minimal viral load

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