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RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in human enterovirus a–d from Clinical Specimens

Biomedical and Environmental Sciences 2020年第11期33卷 829-838页

作者： CHEN Wei WENG Yu Wei HE Wen Xiang ZHU Ying YU Ting Ting XIE Jian Feng ZHENG Kui Cheng YAN Yan Sheng ZHANG Yong Jun ZHANG Wen Chang The School of Public Health Fujian Medical UniversityFuzhou 350122FujianChina Fujian Center for disease Control and Prevention Fujian Provincial Key Laboratory of Zoonosis ResearchFuzhou 350001FujianChina

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 CCId50 perμL and copies perμL,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 CCId50 perμL and 10 virus copies perμL,and for the complete VP1 gene was 1 CCId50 perμL and 100 virus copies perμL,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.

关键词： Clinical specimens human enterovirus a–d VP1 gene Polymerase chain reaction

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