Real-Time RT-PCR in COVID-19 Detection: Introduction, Principle, Procedure and Result Interpretation

Real-Time RT-PCR in COVID-19 Detection: Introduction, Principle, Procedure and Result Interpretation

 Introduction of Real-Time RT-PCR in COVID-19 Detection

SARS-CoV-2 genes detection using real-time RT-PCR is both a screening and confirmatory test of COVID-19 disease and SARS-CoV-2 stands for  Severe acute respiratory syndrome coronavirus. It is a causative agent of  Coronavirus disease 2019 or COVID -19 is a viral disease. It is a positive-sense single-stranded RNA virus and strain was first discovered in Wuhan, China, o it is sometimes referred to as the “Wuhan virus” or “Wuhan coronavirus”. The virus is primarily spread between people through close contact and via respiratory droplets produced from coughs or sneezes. It mainly enters human cells by binding to the receptor angiotensin-converting enzyme 2 (ACE2).

The SARS-CoV-2 nucleic acid test kit is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in upper and lowers respiratory tract specimens such as oropharyngeal and nasopharyngeal swabs and sputum for detection of N gene, ORF1 ab, and human housekeeping gene RNase P.

Principle of SARS-CoV-2 Genes detection using Real-time RT-PCR

By multiplex PCR –fluorescent probe technology combined with fast one-step RT – PCR technology, the kit uses SARS – CoV-2 ORF1 ab and the specific conserved sequence of nucleocapsid protein  N gene as target regions. With the designed dual-target gene detection primer-probe, the kit works with a high-efficiency RT – PCR reaction system for detection. After the end of the reaction, the result will be determined through analysis of the cycle threshold  (Ct) of each channel. Moreover, the kit is designed with the human housekeeping gene RNaseP for monitoring sampling, extraction, specimen addition, amplification, and other related processes as an internal standard to effectively prevent false positive and false negative results, so as to ensure the specificity and accuracy of the test results.

Requirements for SARS-CoV-2 Genes detection using Real-time RT-PCR

Extracted RNA

The SARS-CoV-2 nucleic acid test kit contains-

  • SARS-CoV-2 reaction fluid
  • RT-PCR enzyme
  • SARS-CoV-2 positive quality control
  • Negative quality control
  • Refrigerators
  • Thermocycler (PCR machine )
  • Micropipettes with filter tips
  • PCR cabinet
  • PCR tubes with cap
  • PCR tube holder

Procedure of SARS-CoV-2 Genes detection

All reagents must be thawed completely before use. After mixing centrifuge them at 6000rpm for a few seconds before use.

Specimen extraction
The nucleic acid extraction kit will be used for specimen extraction. The extracted nucleic acid will be tested immediately, or it shall be stored at -20 °C. The storage time shall not exceed three days, and repeated freeze-thaw cycles shall be avoided.

No extraction is required for the kit SARS – CoV-2 positive and negative controls.

Use recommended commercial nucleic acid extraction kit.

Mater Mix Setup

The volume of SARS –CoV-2 reaction fluid and RT- PCR enzyme per reaction multiply with the number of samples, which includes the number of the control and samples prepared. For reasons of unprecise pipetting, always add an extra virtual sample. Mix completely and then spin down briefly with a centrifuge.

Master Mix preparation

Template addition 

Pipet 20 µl Master mix with micropipette of sterile tips to each of the Real-Time PCR reaction plates /tubes. Separately add 5 µl template (negative control and nucleic acid extracted from specimen, positive control) to different reaction plates /tubes. Immediately close the plates/tubes to avoid contamination, centrifuge the mixture instantaneously in order to collect the Master Mix in the bottom of the reaction tubes.

Template addition

RT-PCR amplification

Put the complete PCR reaction tube into the fluorescent quantitative PCR analyzer, set the positive quality control detection hole,  and set the specimen name.

Total reaction system:25 µl

Fluorescence detection channel selection: FAM, HEX, and  ROX  channel are selected for detection: FAM is the N gene indicator channel, HEX  is the ORF1 ab indicator channel, and ROX  is the internal control RNaseP indicator channel. The cycle parameter setting is shown in the table.

Cycle parameter setting

Note-There is no reference fluorescence for the kit, and select “None” for the quencher; save the file after setting, and run the reaction program.

Result analysis for  SARS-CoV-2 Genes detection 

After the reaction is completed, the system saves the results automatically, and the baseline and the threshold of each detection are adjusted according to the image after analysis. Click Analysis for the analysis and make the parameters meet the requirements in  Quality control and then check the result of each unknown specimen.

Quality control

Negative quality control: no exponential increase in ROX channel amplification plot or Ct≥35, no exponential increase in amplification plots of FAM channel and HEX channel or Ct≥ 40.

Positive control: the amplification plots of the three channels, i.e FAM, HEX, and ROX, all show an exponential increase and Ct is <35.

The above two items must be satisfied in the same test at the same time, or the test is deemed invalid and the detection will be carried out again.

Cut–off (CO ) Value or Reference Interval 

Based on the clinical specimen test results the ROC  curve method is adopted to determine the CO value of these kits as follows.

  1. It is FAM positive if the FAM channel amplification plot shows an exponential increase with Ct <40.
  2. It is HEX positive if the HEX channel amplification plot an exponential increase with Ct<40.
  3. It is ROX positive if the HEX channel amplification plot an exponential increase with Ct<35.

INTERPRETATION OF TEST RESULTS for SARS-CoV-2 Genes detection 

  1. In each experiment, it’s necessary to test negative quality control and SARS – CoV-2 positive quality control, and the results can only be determined when the results meet the quality control requirements.
  2. The criterion for a positive specimen: it can be exported as an appositive specimen when the ROX channel is positive and the results of FAM and/or HEX channels are positive.
  3. The criterion for a negative specimen: it can be exported as an appositive specimen when the ROX channel is positive and FAM and HEX channels are negative.
  4. When the ROX channels are negative, the test results of the specimen are invalid. It may be caused by sampling, extraction, specimen addition, amplification, and other processes. It is recommended to conduct the test after pre-extraction or after re-sampling,

Further Reading on Real-Time RT-PCR in COVID-19 Detection

  1. Giaimo C (1 April 2020). “The Spiky Blob Seen Around the World”. The New York Times. Retrieved 6 April 2020.
  2. Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, et al. (March 2020). “The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2”. Nature Microbiology. 5 (4): 536–544. doi:10.1038/s41564-020-0695-z. PMID 32123347. Archived from the original on 5 March 2020. Retrieved 3 March 2020.
  3. “Coronavirus disease named Covid-19”. BBC News Online. 11 February 2020. Archived from the original on 15 February 2020. Retrieved 15 February 2020.
  4. Surveillance case definitions for human infection with the novel coronavirus (nCoV): interim guidance v1, January 2020 (Report).World Health Organization. January 2020. hdl:10665/330376. WHO/2019-nCoV/Surveillance/v2020.1.
  5. “Q&A on coronaviruses (COVID-19)”. World Health Organization (WHO). 11 February 2020. Archived from the original on 20 January 2020. Retrieved 24 February 2020.
  6. “How COVID-19 Spreads”. U.S. Centers for Disease Control and Prevention (CDC). 27 January 2020. Archived from the original on 28 January 2020. Retrieved 29 January 2020.
  7. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200401-sitrep-72-covid-19.pdf?sfvrsn=3dd8971b_2
  8. https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html
  9. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance
  10. file:///C:/Users/Berry/Downloads/WHO-COVID-19-laboratory-2020.5-eng.pdf
  11. Brochure of manufacturer: Guangzhou Wondfo Biotech Co., Ltd
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