单增李斯特菌微滴式数字PCR检测方法的建立及在标准物质研制方面的应用  

Establishment of Droplet Digital PCR Method for Detection of Listeria monocytogenes and Its Application in the Development of Reference Materials

作　　者：徐佳微 辛长卫 李铁山[2] 赵格[1] 曲志娜[1] 赵建梅[1] 高玉斌 张喜悦[1] 王君玮[1] XU Jia-Wei;XIN Chang-Wei;LI Tie-Shan;ZHAO Ge;QU Zhi-Na;ZHAO Jian-Mei;GAO Yu-Bin;ZHANG Xi-Yue;WANG Jun-Wei(Pathogenic Microorganism Monitoring Room,China Animal Health and Epidemiology Center,Qingdao 266032,China;Department of Rehabilitation Medicine,The Affiliated Hospital of Qingdao University,Qingdao 266003,China;Bo'ai County Vocational Secondary Vocational School,Bo'ai 454450,China)

机构地区：[1]中国动物卫生与流行病学中心致病微生物监测室,青岛266032 [2]青岛大学附属医院康复二科,青岛266003 [3]博爱县职业中等专业学校,博爱454450

出　　处：《农业生物技术学报》2024年第10期2413-2423,共11页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2022YFC2303900,2022YFD1301003);中国动物卫生与流行病学中心创新基金(DW2021001-13)。

摘　　要：单核细胞增生李斯特菌(Listeria monocytogenes)是最严重的食源性病原菌之一。本研究以细胞壁水解酶基因(invasion associated protein,iap)为靶基因建立了微滴式数字PCR(droplet digital PCR,ddPCR)方法,优化了反应条件并测试了方法的特异性、灵敏度和重复性,并利用所建立的方法进行了临床样品的检测以及应用该方法研制其标准物质。结果显示,在10μmol/L引物用量为1.5μL、10μmol/L探针用量为0.45μL和退火温度在60℃时,ddPCR方法的扩增效果最好,最终成功建立了单增李斯特菌ddPCR检测方法。所建立的ddPCR检测方法具有较好的特异性,与其他非特异性菌株不发生交叉反应;重复性试验结果显示组间变异系数为1.57%~4.32%,证实该方法重复性好;该方法具有较高的灵敏度,最低检出下限为6.65 copies/μL。利用ddPCR方法和qPCR方法检测实验室保存的47份临床阳性样本,符合度为100%;应用所建立的方法对标准物质进行均匀性和稳定性检验,并联合9家实验室对标准物质定值,定值结果为5.79×10^(3) copies/μL,不确定度为0.47×10^(3) copies/μL。本研究建立的ddPCR检测方法可用于实验室中单增李斯特菌的检测和标准物质的研制,可成为监测单增李斯特菌感染的一种技术手段。Listeria monocytogenes is one of the most serious foodborne pathogens.The aim of this study is to establish a rapid,sensitive,and accurate droplet digital PCR(ddPCR)method for detecting L.monocytogenes,and apply the method to develop the reference materials.In the study,the ddPCR method was established with the invasion associated protein gene(iap)as the target gene,the reaction conditions were optimized,the specificity,sensitivity,and repeatability of the method were tested,and the established method was used to detect clinical samples and the standard material was developed by using the method.The results showed that the ddPCR method had the best amplification effect when the amount of 10μmol/L primer was 1.5μL,the amount of 10μmol/L probe was 0.45μL and the annealing temperature was 60℃.Finally,the ddPCR method for L.monocytogenes was established.The established ddPCR method had good specificity and did not crossreact with other non-specific strains.Repeatability test results showed that the coefficient of variation between groups was 1.57%~4.32%,which proved that the method had good repeatability.The method had high sensitivity,and the lower limit of detection was 6.65 copies/μL.The results of clinical samples showed that the ddPCR method and fluorescence quantitative PCR method were 100% consistent with the results of 47 clinical positive samples stored in the laboratory.This method was used to measure the uniformity and stability of the reference materials,and 9 laboratories determined the reference materials.The fixed value was 5.79×10^(3) copies/μL,and the uncertainty was 0.47×10^(3) copies/μL.The ddPCR method established in this study can be used for the detection of L.monocytogenes in the laboratory and the development of reference materials,etc.,and can be a technical means to monitor L.monocytogenes infection.

关 键 词：单核细胞增生李斯特菌 微滴式数字PCR(ddPCR) iap基因 标准物质 

分 类 号：S852.61[农业科学—基础兽医学] Q93-331[农业科学—兽医学]