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SARS-CoV-2 PCR Assays

The SARS-CoV-2 virion contains four proteins and single stranded RNA. The four proteins include spike protein, envelope protein, membrane protein and nucleocapsid. The RNA genome consists of 29,900 nucleotides which is larger than most other RNA viruses. One third of the genome consists of genes for four structural proteins and eight genes for accessory proteins that inhibit host defenses. Most of the remainder of the genome consists of the replicase gene. The replicase gene codes for two large polyproteins that are cleaved into 15 or 16 nonstructural proteins (NSP) and assist in replicating and proofreading the viral genome.

Assays used in many laboratories across Canada and internationally are real time PCR assays targeting two different amplification regions, the E (envelope protein) and RdRp (RNA-dependent RNA polymerase) genes. The real time PCR designed by the United States Centers for Disease Control (CDC uses three different amplification regions. NS3 was designed for universal detection of SARS-like coronaviruses, while the N1 and N2 regions are specific for SARS-CoV-2. The NS3 target produced too many false positive results and had to be eliminated.

Recent studies indicate that viral load peaks in the first week of disease onset. Viral RNA can be detected during the secondweek of disease onset, but viral load is low. RT-PCR is positive in some asymptomatic cases and in recovering patients. One small study suggested that some patients may continue to be viral carriers after apparent recovery. Four Chinese medical professionals recovered from COVID19 and met the criteria for hospital discharge or discontinuation of quarantine. These criteria included absence of clinical symptoms, resolution of radiological abnormalities and 2 negative RT-PCR test results. All these patients had repeat positive RT-PCR test results 5 to 13 days later.

Despite high sensitivity, a negative PCR is insufficient to exclude SARS-CoV-2 infection in patients with high clinical suspicion. According the interim guidance from the WHO, a single negative test result does not exclude infection with SARS-CoV-2. Patients with a typical clinical presentation or clear epidemic indications should receive clinical treatment and case management, even if PCR is negative at one or two time points. The time of sample collection, quality of the sample, assay performance, quality controls and training of testing professionals all contribute to the accuracy of the testing. Repeat testing using a lower respiratory sample is strongly recommended in severe or progressive disease.

PCR should be an integral part of the routine diagnostic work up of SARS-CoV-2, especially in non-endemic areas. However, if the pretest probability is very high due to high disease prevalence and if many cases of the disease have already been confirmed by NAT in an endemic area, then there is little utility to requiring laboratory or radiological confirmation of the disease. This approach is similar to CDC recommendations for influenza testing in the U.S.

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