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

Introduction: Reverse transcriptase -polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charit & eacute;-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and dela-ying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and RNase P genes. Methods: We compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and RNase 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 = 0.84). 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 Charite, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples. (c) 2021 The Authors. Published by Elsevier Espana,

Automated summary

This study validates the effectiveness of duplex PCR technique (E gene and RNase P gene) in COVID-19 diagnosis and demonstrates that it does not affect the results reported by the Charité, Berlin protocol. Duplex PCR is a useful tool for screening clinical samples.