Development of a rapid LFA test based on direct RT-LAMP for diagnosis of SARS-CoV-2.

Introduction: In response to the rapid spread of the SARS-CoV-2 virus, we developed a rapid molecular approach to diagnose COVID-19 without the need for RNA extraction.

Methods: The study utilized two molecular methods, RT-qPCR and colorimetric RT-LAMP, to diagnose the RdRp and ORF8 genes, respectively, in oro-nasopharyngeal swabs. Due to the high sequence diversity of ORF8 in SARS-CoV and SARS-CoV-2, it has been identified as a suitable target for virus detection. The RT-LAMP method was also carried out directly on heat-treated swab samples. The strip tests were made using gold nanoparticles and combined with the RT-LAMP for further analysis.

Results: The results showed that the isothermal amplification method had a sensitivity of 95 % (95 % C.I.: 86.08 %-98.96 %) and a specificity of 75 % (95 % C.I.: 19.41 %-99.37 %). The RT-LAMP-LFA method was able to distinguish positive and negative samples with 100 % sensitivity (95 % C.I.: 91.96-100) and 77.27 % specificity (95 % C.I.: 54.63-92.18). This method only required heating swab samples for 10 min at 65 °C before the RT-LAMP reaction.

Conclusion: By utilizing the RT-LAMP in combination with the LFA, it is possible to diagnose SARS-CoV-2 rapidly without the need for RNA extraction. The entire process from sample collection to test interpretation takes only 75-90 min, and the results can be interpreted by untrained individuals with the naked eye. By employing the ORF8 gene as a diagnostic target and eliminating the need for RNA extraction, the direct RT-LAMP-LFA method achieves a significant breakthrough that was not previously reported.