建立双重荧光定量PCR方法检测蜱传疏螺旋体  

Establishing a duplex real-time PCR method for the detection of tick-borne Borrelia

作　　者：杨小娜 张琳[1] 侯学霞[1] 郝琴[1] YANG Xiao-na;ZHANG Lin;HOU Xue-xia;HAO Qin(State Key Laboratory for Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206

出　　处：《中国人兽共患病学报》2021年第9期788-794,共7页Chinese Journal of Zoonoses

基　　金：“十三五”国家重大科技专项(No.2017ZX10303404-006-003)

摘　　要：目的建立双重荧光定量PCR方法,同时检测Borrelia burgdorferi和Borrelia miyamotoi。方法分别利用recA基因、glpQ基因合成引物探针,并优化反应条件。在单重实验的基础上建立双重荧光定量PCR方法,对该方法灵敏度和特异度进行检测,并探索其他因素对反应结果的影响。使用模拟样本和实际样本评价新方法的检测效用。结果双重荧光定量PCR方法具有良好的灵敏度和特异度,与单重实验方法比较,检测Ct值无明显差异(P>0.05),病原浓度与检测Ct值具有良好的线性相关关系。两种病原浓度差异和其他病原的存在对检测结果无明显影响。使用该方法检测模拟蜱样本和实际蜱样本具有良好的实用性。结论本实验建立的多重荧光定量PCR方法能快速便捷的检测两种疏螺旋体,为蜱样本病原鉴定提供了有价值的检测工具。Borrelia burgdorferi and Borrelia miyamotoi are two pathogens transmitted by hard ticks and can cause diseases in human.Duplex real-time PCR method for simultaneous detection of those two pathogens was established on the basis of singleplex PCR.Primers and probes were designed according to the recA gene of B.burgdorferi and glpQ gene of B.miyamotoi.Duplex real-time PCR also showed good sensitivity and specificity.In the dilution series ranging from 109-102,there was no significant difference between duplex and singleplex PCR in terms of Ct value(P>0.05).Moreover,there was a good linear correlation between pathogen concentration and Ct value.The change in concentration and the presence of other pathogens had no significant influence on the results.The method had good practicability in detecting simulated and actual tick samples.In conclusion,the duplex real-time PCR method established in this study can detect two pathogens simultaneously,and provides a valuable tool for pathogen identification of tick samples.

关 键 词：伯氏疏螺旋体 Borrelia miyamotoi 双重荧光定量PCR 

分 类 号：R377[医药卫生—病原生物学]