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SARS-CoV-22 (COVID-19) Testing: Status Update 11 April 2020 9:00 PM IST
A total of 1,79,374 samples from 1,64,773 individuals have been tested as on 11 April 2020, 9 PM
IST. 7703 individuals have been confirmed positive among suspected cases and contacts of known
positive cases in India.
Today, on 11 April 2020, till 9 PM IST, 17,143 samples have been reported. Of these, 600 were
positive for SARS-CoV-2.
Around 154 government institutions and 68 private institutions are running for assuring the proper testing of suspected patients
Dates: 11 April, 2020.
The Indian council of medical research has enforced a rapid action team.
https://media.defense.gov/2011/Oct/16/2000209538/-1/-1/0/110921-F-BE781-030.JPG.
India is using this method of detection of coronavirus suspects.
Multiplex Real-Time PCR for detection of SARS-CoV-2 using TaqPath
COVID-19 Combo Kit (Applied Biosystems).
1. Introduction:
The purpose of this document is to provide interim guidance to laboratories involved in laboratory testing of
patients who meet the definition of a suspected case of pneumonia associated with a novel coronavirus identified
in Wuhan, China.
TaqPath™ COVID-19 Combo Kit contains the assays and controls for a real-time reverse transcription
polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from SARS-
CoV-2 in the nasopharyngeal swab, nasopharyngeal aspirate, and bronchoalveolar lavage (BAL) specimens from
individuals suspected of COVID-19 by their healthcare provider. TaqPath™ COVID-19 Combo Kit is for use
only under Emergency Use Authorization (EUA).2. SourceReference:
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0019181_TaqPath_COVID-
19_IFU_EUA.pdf
3. Testing criteria/Objective:
Detection of SARS-CoV-2 in human clinical specimens using TaqPath COVID-19 Combo Kit (Applied
Biosystems)
4. Principle:
The real-time assay uses the TaqMan fluorogenic probe-based chemistry that uses the 5´ nuclease activity of
Taq DNA polymerase and enables the detection of a specific PCR product as it accumulates during PCR
cycles.
COVID-19 Real-Time PCR Assay Multiplex-Multiplexed assays that contain three primer/probe sets specific
to different SARS-CoV-2 genomic regions and primers/probes for phage MS2 (Internal process control for
nucleic acid extraction).
5. Safety procedures: According to the Laboratory Safety Manual. (WHO,2011)
6. Sample requirements: 250μl of the specimen or as per the recommended kit.
7. Standard and controls:
(1) Positive control (Supplied with it)
8. Scope and definition:
Highly specific and efficient detection of SARS-CoV-2 by Multiplex Real-Time PCR.
9. Requirements:
Equipment Consumables Reagents and samples
Water bath, Bio Safety Cabinet
clean laminar flow hood with
micro-centrifuge with plate rotor
and vortex, MiniSpin. Pipette set,
Real-Time PCR machine.
Mask, gloves, Lab Coats sterile filter
tips, tissue paper, 0.2 ml, 0.5 ml and
1.5 ml microcentrifuge tubes, micro
tips, 0.5-10 µl, 20-200 µl, and 1000 µl
tips. Real-Time PCR Plates and
sealers or tubes and strips
TaqPath COVID-19 Combo Kit
(Applied Biosystems), Milli Q
Water, Extracted viral Nucleic
samples,
10. test procedure:
1. *Add 10 µL MS2 Phage Control to each sample well and the Negative Control well during extraction
and perform RNA extraction of clinical specimens using your laboratory protocol. Extracted RNA will be
the starting point for the reaction.
2. Prepare real-time PCR worksheets (KGMU- VIRO-RTM-MPX-PCR-PP-6.7-copy attached at the end)
3. Perform multiplex real-time PCR reaction as shown in table1 for corona ORF1ab gene, N gene, S gene
and MS2 (Internal process control for nucleic acid extraction) in a single tube (as per manufacturers
instruction).
4. Determine the number of reactions (N) to set upper assay. Also, include Negative control &
Positive control in the test.
5. Prepare excess reaction cocktail to account for pipetting error.
If number of samples (n) including controls = 1 to 10, then N = n + 2
6. In the clean reagent preparation room, prepare the MasterMix:
Calculate the amount of each reagent to be added for each set reaction master mix.
Table 1: The calculations are as follows:
S.No. Component Volume for one
reaction(N=1)
Volume(N= )
1. TaqPath™ 1-Step Multiplex
MasterMix(NoROX™)(4X)
6.25 µL
2. COVID-19 Real-Time PCR
Assay Multiplex
1.25 µL
3. Nuclease-free Water 12.50 µL
Total Reaction Mix volume 20.0 µL
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