A Comparative Analysis of Molecular Biological Methods for the Detection of SARS-CoV-2 and Testing the In Vitro Infectivity of the Virus

Abstract

The virus discovered in 2019 in the city of Wuhan, China, which was later identified as SARS-CoV-2 and which spread to the level of a pandemic, put diagnostic methods to the test. Early in the pandemic, we developed a nested PCR assay for the detection of SARS-CoV-2, which we validated and applied to detect the virus in feline samples. The present study describes the application of the nested PCR test in parallel with LAMP for the detection of the virus in 427 nasopharyngeal and oropharyngeal human samples taken between October 2020 and January 2022. Of the swabs tested, there were 43 positives, accounting for 10.1% of all samples tested, with the negatives numbering 382, i.e., 89.5%, and there were 2 (0.4%) invalid ones. The nPCR results confirmed those obtained by using LAMP, with results concordant in both methods. Nasal swabs tested using nPCR confirmed the results of oropharyngeal and nasopharyngeal swab samples tested using LAMP and nPCR. The focus of the discussion is on the two techniques: the actual practical application of the laboratory-developed assays and the diagnostic value of nasal samples. The nPCR used is a reliable and sensitive technique for the detection of SARS-CoV-2 in nasopharyngeal, oropharyngeal, and nasal swab samples. However, it has some disadvantages related to the duration of the entire process, as well as a risk of contamination. Experiments were performed to demonstrate the infectivity of the virus from the positive isolates in vitro. A discrepancy was reported between direct and indirect methods of testing the virus and accounting for its ability to cause infection in vitro.

Keywords

• nested PCR
• LAMP
• nasal swabs
• SARS-CoV-2
• diagnostic
• IVDR