RESUMO
Emergence of new variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) during current pandemic of Coronavirus Disease 2019 (COVID-19) and several waves of infections by some of variants emphasized the importance of continuous surveillance. While genomic surveillance through whole genome sequencing is performed as a standard method, identification of known variants through mutation-targeting molecular diagnosis such as qRT-PCR is also useful for timely investigation. However, there are limited studies regarding the concurrent detection and identification of SARS-CoV-2 variants through a LAMP-based method. In this study, we developed and evaluated RT-LAMP assays to detect characteristic deletions of SARS-CoV-2 variants. In addition, we evaluated a fluorescent probe mediated method for identification of single nucleotide substitution by RT-LAMP. Finally, we discussed restrictions and perspectives regarding pathogen screening and surveillance of variants by RT-LAMP based on our observations.
RESUMO
The World Health Organization (WHO) has declared the Coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of viral RNA by RT-PCR significantly varied according to the sequence of primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be significantly higher than RT-qPCR. These findings suggest that ddPCR could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.
RESUMO
Epidemics of Coronavirus Disease 2019 (COVID-19) now have more than 100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by RT-qPCR methods, but the capacity of RT-qPCR methods is limited by its requirement of high-level facilities and instruments. Here, we developed and evaluated RT-LAMP assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human Coronaviruses was not observed. We also adapted a colorimetric detection method for our RT-LAMP assay so that the tests potentially performed in higher throughput.
RESUMO
Coronavirus disease 2019 (COVID-19) is newly emerging human infectious diseases, which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within two months of the outbreak, more than 80,000 cases of COVID-19 have been confirmed worldwide. Since the human to human transmission occurred easily and the human infection is rapidly increasing, the sensitive and early diagnosis is essential to prevent the global outbreak. Recently, World Health Organization (WHO) announced various primer and probe sets for SARS-CoV-2 previously developed in China, Germany, Hong Kong, Japan, Thailand, and USA. In this study, we compared the ability to detect SARS-CoV-2 RNA among the seven primer-probe sets for N gene and the three primer-probe sets for Orf1 gene. The result of the comparative analysis represented that the 2019-nCoV_N2, N3 of USA and the ORF1ab of China are the most sensitive primer-probe sets for N and Orf1 genes, respectively. Therefore, the appropriate combination from ORF1ab (China), 2019-nCoV_N2, N3 (USA), and NIID_2019-nCOV_N (Japan) sets should be selected for the sensitive and reliable laboratory confirmation of SARS-CoV-2.
