鼠疫多重荧光定量PCR内标检测方法的建立与应用评价  

Establishment and evaluation of internal control of multiplex quantitative PCR for detection of Yersinia pestis

作　　者：李博[1] 崔燕[1] 安静[2] 古丽阿依·包开西[1] 罗勇军 王信惠[1] 黎唯[1] 王效俊[1] Li Bo;Cui Yan;An Jing;Guliayi·Baokaixi;Luo Yongjun;Wang Xinhui;Li Wei;Wang Xiaojun(Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention,Urumqi 830001,Xinjiang,China;Xinjiang Academy of Agricultural Sciences,Urumqi 830091,Xinjiang,China)

机构地区：[1]新疆维吾尔自治区疾病预防控制中心,新疆乌鲁木齐830001 [2]新疆农业科学院,新疆乌鲁木齐830091

出　　处：《疾病监测》2022年第5期657-660,共4页Disease Surveillance

基　　金：新疆维吾尔自治区自然科学基金(No.2018D01C089)。

摘　　要：目的建立一种鼠疫多重荧光定量PCR内标检测方法,并进行应用效果评价。方法选择鼠疫菌染色体YPO0393基因与pMT1质粒caf1基因为靶基因,设计合成YPO0393-内标模板、特异性引物和TaqMan荧光探针,构建鼠疫多重荧光定量PCR内标检测体系;对非鼠疫菌DNA、不同稀释浓度鼠疫菌DNA、野外监测样本进行检测,评价该方法的特异性、灵敏性、重复性和应用效果。结果采用多重荧光定量PCR内标方法,8株鼠疫菌DNA均出现明显的扩增曲线,12株非鼠疫菌DNA未显示扩增曲线,提示该方法特异性良好。不同浓度的鼠疫EV76疫苗株DNA,检测最低限为22.5×10^(-4)ng/μL,变异系数为0.99~3.42,提示灵敏性、重复性较好。105份野外监测样本中,荧光定量PCR内标方法检测阳性的6份,与普通PCR方法检测结果一致,4份样本分离培养出鼠疫菌且采用PCR方法检测阳性。结论通过设置双靶基因与内标对照,本研究建立的鼠疫多重荧光定量PCR内标方法可降低检测假阳性率,特异性、灵敏性和重复性理想,可用于鼠疫快速检测。Objective To establish an internal control of multiplex quantitative PCR for the detection of Yersinia pestis and evaluate its effect.Methods A multiplex quantitative PCR system was established using the YPO0393 gene of the chromosome of Y.pestis and caf1 gene of plasmid pMT1 as the target gene.The internal control was YPO0393.Specific primers and probes were designed.Detection was performed for DNA of the control,Y.pestis and field samples.The specificity,sensitivity and repeatability of this assay were evaluated.Results All the 8 DNA samples of Y.pestis at different concentrations showed significant S-curve amplification.The DNA of 12 controls showed no amplification curve,suggesting that the multiplex quantitative PCR was specific.The detection sensitivity of the multiplex quantitative PCR was 22.5×10^(-4)ng/μL,and the coefficient of variation ranged from 0.99 to 3.42 for the EV76 strain of Y.pestis,indicating good sensitivity and repeatability.In the 105 field samples,6 were determined as positive by the multiplex quantitative PCR,which was consistent with routine PCR.Y.pestis was isolated from 4 positive field samples.Conclusion The multiplex quantitative PCR has high specificity,sensitivity and repeatability by using the YPO0393 gene as internal control,by which the false negative rate can be reduced.Therefore,this assay can be applied in the rapid detection of Yersinia pestis.

关 键 词：鼠疫耶尔森菌 多重荧光定量PCR方法 内标对照 评价 

分 类 号：R211[医药卫生—中医学] R516.8