Development and Optimization of Classic Multiplex RT-PCR for the SARS-CoV-2 Detection

Abstract

Develop alternative methods for the identification of SARS-CoV-2 is essential for epidemiologic studies and interspecies correlations, allowing the allocation of scant resources to the Real-Time PCR (qRT-PCR) for patient diagnosis. The aim of this study, therefore, was to develop and optimize a multiplex reverse transcription-PCR (RT-PCR) assay to detect SARS-CoV-2 in nasopharynx clinical samples as an alternative to qRT-PCR. Bioinformatics tools to specific primers design analyzed genome sequences of the new coronavirus available on GenBank. The assay was proposed to amplify partial segments of N genes and RdRp region of the orflab gene of SARS-CoV-2, recommended targets by the World Health Organization (WHO). Reaction control used positive commercial control and samples from patients known to be positive and negative. The results of this study demonstrated that it was possible to optimize the RT-PCR for the detection of SARS-CoV-2 through a multiplex assay in agreement with the gold standard, precursors results for validation studies of this alternative method for epidemiological and animal health surveys are until now unclear.