基于多重PCR的呼吸道病毒纳米孔测序快速检测的方法学研究  

Rapid detection of respiratory viruses by nanopore sequencing based on multiplex PCR:A methodological study

作　　者：申业壮 刘冉 尹晓尧 胡曼东 张雅薇 何昆[1] 周涛[1] 李卫华[1] 解现星 SHEN Yezhuang;LIU Ran;YIN Xiaoyao;HU Mandong;ZHANG Yawei;HE Kun;ZHOU Tao;LI Weihua*;XIE Xianxing(National Center of Biomedical Analysis,Beijing 100850,China)

机构地区：[1]国家生物医学分析中心,北京100850

出　　处：《军事医学》2023年第11期823-828,共6页Military Medical Sciences

摘　　要：目的通过对多重PCR扩增条件的优化和纳米孔测序中病原微生物检测特异性及灵敏度的评价,建立一种由多重PCR和纳米孔测序技术相结合的检测技术体系,满足对呼吸道病毒现场快速检测鉴定的需求。方法首先针对呼吸道病毒设计特异性引物,其次优化PCR扩增条件,建立快速测序体系,最后对不同浓度的病原体进行扩增与测序鉴定,评价检测体系的灵敏度。结果优化后的多重PCR检测体系在退火温度为55℃、引物添加浓度为0.1µmol/L时,可同时检出25种呼吸道病毒,检测灵敏度可达1×104拷贝/ml,整体检测时间在4 h以内。结论该方法简单快捷,能够实时获取扩增片段的序列信息,特异性强,灵敏度较高,有望为呼吸道病原体的快速筛选和现场检测鉴定提供有力技术支撑。Objective To establish a detection technology system that combines multiplex PCR and nanopore sequencing to meet the needs of on⁃site rapid detection and identification of respiratory viruses.Methods Specific primers were designed to amplify respiratory viruses before PCR amplification conditions were optimized and a rapid sequencing system was established,followed by analysis of the data and evaluation of pathogen detection sensitivity.Results The optimized multiplex PCR amplifi⁃cation conditions included an annealing temperature of 55℃and primer concentration of 0.1µmol/L.Using this system,25 respiratory viruses could be detected simultaneously.The detection sensitivity reached 1×104 copies/ml,and detection generally took less than 4 h.Conclusion This detection method is simple and fast,and capable of obtaining sequence information of amplified fragments in real time with a specificity and sensitivity.It is likely to provide strong technical support for the rapid screening,on⁃site detection,and identification of respiratory pathogens.

关 键 词：呼吸道病毒 多重PCR 纳米孔测序 快速检测 特异性 

分 类 号：R373[医药卫生—病原生物学]