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Updated: Nov 11, 2025
EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR
Published on: January 16, 2016
RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens
Wei Chen1, Yu Wei Weng2, Wen Xiang He2
1The School of Public Health, Fujian Medical University, Fuzhou 350122, Fujian, China.
Objective:
To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.
Methods:
A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID 50 per μL and copies per μL, and the newly established methods were tested in clinical specimens collected in recent years.
Results:
The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID 50 per μL and 10 virus copies per μL, and for the complete VP1 gene was 1 CCID 50 per μL and 100 virus copies per μL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons.
Conclusion:
This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.
