6种常见鼠传病原体双重实时荧光定量PCR检测方法构建  

Biplex quantitative real-time PCR for detection of six common rodent-borne pathogens

作　　者：陈敏 梁莹[1] 刘起勇[1] 栗冬梅[1] CHEN Min;LIANG Ying;LIU Qi-yong;LI Dong-mei(Department of Vector Biology 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媒介生物控制室,北京102206

出　　处：《中国媒介生物学及控制杂志》2025年第1期99-105,共7页Chinese Journal of Vector Biology and Control

基　　金：媒介生物监测与控制项目(102393220020020000012)。

摘　　要：目的建立双重实时荧光定量PCR(qPCR)方法,用于土拉弗朗西斯菌、巴尔通体、问号钩端螺旋体、地方性斑疹伤寒立克次体、嗜吞噬细胞无形体及恙虫病东方体等6种鼠传病原体的快速检测。方法根据已报道的qPCR方法,建立3种双重qPCR方法,分别检测土拉弗朗西斯菌和恙虫病东方体、巴尔通体和问号钩端螺旋体、地方性斑疹伤寒立克次体和嗜吞噬细胞无形体。绘制标准曲线,并对其灵敏度、特异性和重复性进行检测。同时,对鼠组织核酸模拟标本和阳性标本进行检测,以验证方法的可靠性。结果建立的3种双重qPCR方法均只扩增目的病原体,其他病原体和阴性对照均未出现荧光信号;6种病原体最低检测下限均为1×10^(1)拷贝/μl;循环阈值(Ct)变异系数为0.04%~1.20%;对核酸模拟标本和阳性标本进行检测,结果全为阳性,一致性100%。结论建立的双重qPCR方法特异性好、敏感性高、重复性好,可用于快速、准确检测土拉弗朗西斯菌、巴尔通体、问号钩端螺旋体、地方性斑疹伤寒立克次体、嗜吞噬细胞无形体及恙虫病东方体。Objective To establish biplex quantitative real-time polymerase chain reaction(qPCR)methods for rapid detection of six rodent-borne pathogens,including Francisella tularensis,Bartonella spp.,Leptospira interrogans,Rickettsia typhi,Anaplasma phagocytophilum,and Orientia tsutsugamushi.Methods Three biplex qPCR methods were established based on the reported qPCR assays for detection of F.tularensis and O.tsutsugamushi,Bartonella spp.and L.interrogans,R.typhi and A.phagocytophilum,respectively.Standard curves were plotted,and the sensitivity,specificity,and repeatability of these methods were determined.Meanwhile,simulated nucleic acid samples of rodent tissues infected with six rodent-borne pathogens and positive samples were detected to validate the reliability of the method.Results The three established biplex qPCR methods amplified only the target pathogens,and other pathogens and negative controls did not show fluorescence signals.The lower limit of detection for all six pathogens was 1×10^(1)copies/μl;the coefficient of variation of cycle threshold values ranged from 0.04%to 1.20%.The results of the simulated and positive samples were positive,with 100%consistency.Conclusion These biplex qPCR methods have good specificity,high sensitivity,and good repeatability,which can be used to rapidly and accurately detect F.tularensis,Bartonella spp.,L.interrogans,R.typhi,A.phagocytophilum,and O.tsutsugamushi.

关 键 词：鼠传病原体 双重实时荧光定量PCR 检测 

分 类 号：R37[医药卫生—病原生物学]