COVID-19 Diagnostic RT-PCR Protocol

The 2019-nCoV laboratory test assays were based on the previous WHO recommendation.¹⁰ Upper and lower respiratory tract specimens were obtained from patients. RNA was extracted and tested by real-time RT-PCR with 2019-nCoV–specific primers and probes. Tests were carried out in biosafety level 2 facilities at the Hubei (provincial) CDC and then at the National Institute for Viral Disease Control at China CDC. If two targets (open reading frame 1a or 1b, nucleocapsid protein) tested positive by specific real-time RT-PCR, the case would be considered to be laboratory-confirmed. A cycle threshold value (Ct-value) less than 37 was defined as a positive test, and a Ct-value of 40 or more was defined as a negative test. A medium load, defined as a Ct-value of 37 to less than 40, required confirmation by retesting. If the repeated Ct-value was less than 40 and an obvious peak was observed, or if the repeated Ct-value was less than 37, the retest was deemed positive. The genome was identified in samples of bronchoalveolar-lavage fluid from the patient by one of three methods: Sanger sequencing, Illumina sequencing, or nanopore sequencing. Respiratory specimens were inoculated in cells for viral isolation in enhanced biosafety laboratory 3 facilities at the China CDC.^{3 (link)}