End-point RT-PCR: A potential alternative for diagnosing coronavirus disease 2019 (COVID-19)

Real-time reverse transcription-polymerase chain reaction (RT-qPCR) is considered the gold standard for the direct diagnosis of SARS-CoV-2 infections. However, routine diagnosis by RT-qPCR is a limitation for many laboratories, mainly due to the infrastructure and/or disproportionate relationship between demand and supply of inputs. In this context, and to increase the diagnostic coverage of SARS-CoV-2 infections, we describe an alternative, sensitive and specific one-step end-point RT-PCR for the detection of the SARS-CoV-2 E gene. The performance of the RT-PCR was evaluated in 43 clinical samples, of which 10 and 33 were previously identified as negative and positive, respectively, by RT-qPCR. Among the positive samples, 15 and 18 were from asymptomatic and symptomatic individuals, respectively. Here, 32/33 of the positive samples in the RT-qPCR, including from asymptomatic individuals, were found positive in the RT-PCR (Ct 15.94-34.92). The analytical sensitivity of the assay was about 7.15-9 copies of vRNA/mu L, and nonspecific amplifications were not observed in SARS-CoV-2 negative samples. Importantly, the RT-PCR reactions were performed in a 10 mu L final volume. Finally, considering specificity, analytical sensitivity and cost reduction, we believe that the RT-PCR platform described here may be a viable option for the diagnostic of SARS-CoV-2 infections in laboratories in which RTqPCR is not available.

Automated summary

Real-time reverse transcription-polymerase chain reaction (RT-qPCR) is considered the gold standard for diagnosing SARS-CoV-2 infections, but its limitations in many laboratories have led to the development of an alternative, sensitive RT-PCR platform with comparable specificity and sensitivity in a smaller reaction volume. This platform may provide a viable option for SARS-CoV-2 diagnosis in laboratories where RT-qPCR is not available, offering cost efficiency and reliable results.