多重PCR和纳米孔测序结合检测消化道病原体方法及其评价

Multiplex PCR and nanopore sequencing combined to detect gastrointestinal pathogens and its evaluation

目的建立多重PCR和纳米孔测序相结合的检测方法,为实现消化道病原体现场高灵敏度快速检测奠定基础。方法通过设计消化道病原体特异性引物,验证引物特异性,优化多重PCR扩增条件,构建快速建库测序体系,对病原体的检测灵敏度进行评价。结果建立的12种消化道病原体多重PCR检测体系可以对所有靶序列进行有效扩增。经测序检测,当引物池中每条引物浓度为0.1μmol/L时,12种病原体混合DNA模板和3种轮状病毒A、B、C的混合RNA样品检测灵敏度均可达到103拷贝/ml。结论该方法可在短时间内完成消化道病原体的鉴定,提升突发公共卫生事件中现场病原体检测效率,有望在疫情防控中发挥重要作用。

Objective To establish a detection method that combines multiplex PCR and nanopore sequencing for high sensitivity and rapid detection of gastrointestinal pathogens on the scene.Methods Specific primers for gastrointestinal pathogens were designed before the specificity of the primers was verified and multiplex PCR amplification conditions were optimized in order to build a rapid library sequencing system and evaluate the sensitivity of detection.Results The established multiplex PCR detection system for twelve gastrointestinal pathogens could effectively amplify all the target sequences.The sensitivity of detection of the templates with mixed DNA from the twelve pathogens and the mixed RNA samples of three rotavirus A,B and C could reach 103 copies/ml when the concentration of each primer in the primer pool was 0.1μmol/L.Conclusion With this method,gastrointestinal pathogens can be identified in a short time so that the efficiency of on⁃site detection of pathogens in public health emergencies can be improved.This method is expected to play an important role in disease control and prevention.