Validation of a duplex PCR technique using the gen E and RNase P for the diagnosis of SARS-CoV-2.

ABSTRACT

INTRODUCTION: Reverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and ribonuclease P genes. METHODS: We compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and Rnasa P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values ​​(Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. RESULTS: There were no significant differences in the Ct results between both techniques (P = .84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. CONCLUSIONS: Duplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.