ABSTRACT
The emerging pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) critically challenges early and accurate virus diagnoses. However, the current gold standard for SARS-CoV-2 detection, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), has reportedly failed to detect low-viral loads. One compound, graphene oxide (GO), which adsorbs single-stranded DNA (ssDNA), has been widely applied in molecular pathogen detection. This study presents a highly sensitive GO-multiplex qPCR method for simultaneous detection of two SARS-CoV-2 genes (RdRP and E) and one reference gene (RNase P). In a GO-multiplex qPCR system, GO pre-absorbs each forward primer to form specific GO-forward primer composites before entering the amplification system. Target gene amplification is confined within the primer-enriched composites, thus, improving the sensitivity of the assay. Compared to conventional multiplex qPCR, GO-multiplex qPCR reduces the limit of detection by 10-fold to 10 copies/reaction. Hence, the GO-multiplex qPCR assay can be effectively used for SARS-CoV-2 detection.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19 Testing , Clinical Laboratory Techniques/methods , COVID-19/diagnosis , RNA, Viral/genetics , RNA, Viral/analysis , Sensitivity and SpecificityABSTRACT
The emerging pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) critically challenges early and accurate virus diagnoses. However, the current gold standard for SARS-CoV-2 detection, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), has reportedly failed to detect low-viral loads. One compound, graphene oxide (GO), which adsorbs single-stranded DNA (ssDNA), has been widely applied in molecular pathogen detection. This study presents a highly sensitive GO-multiplex qPCR method for simultaneous detection of two SARS-CoV-2 genes (RdRP and E) and one reference gene (the human RNase P). In a GO-multiplex qPCR system, GO pre-absorbs each forward primer to form specific GO-forward primer composites before entering the amplification system. Target gene amplification is confined within the primer-enriched composites, thus, improving the sensitivity of the assay. Compared to conventional multiplex qPCR, GO-multiplex qPCR reduces the limit of detection by 10-fold to 10 copies/reaction. Hence, the GO-multiplex qPCR assay can be effectively used for SARS-CoV-2 detection. Graphical Image 1