RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A–D from Clinical Specimens

• 作者: CHEN Wei HE Wen Xiang WENG Yu Wei XIE Jian Feng YAN Yan Sheng YU Ting Ting ZHANG Wen Chang ZHANG Yong Jun ZHENG Kui Cheng ZHU Ying
• 来源期刊: 生物医学与环境科学（英文版）
• 年，卷（期）: 2020,（11）
• 所属期刊栏目: Original Articles
• 页码范围: 829-838
• 页数: 10页
• 语种: 英文
• DOI: 10.3967/bes2020.113

摘要

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 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 CCID50 per μL and 10 virus copies per μL, and for the complete VP1 gene was 1 CCID50 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.