摘要
产气荚膜梭菌(Clostridium perfringens)是一种重要的人兽共患病原菌,α-毒素是其分子生物学检测的主要靶标。试验选取Clper引物及探针建立微滴式数字PCR(droplet digtial PCR,ddPCR)方法,最佳引物浓度0.85μmol/L,探针浓度为0.3μmol/L,退火温度为60℃。利用建立的ddPCR方法检测单核细胞增生李斯特菌、沙门氏菌、大肠杆菌、肠球菌、金黄色葡萄球菌、弯曲杆菌的DNA,结果均为阴性,表明该方法具有较好的特异性。合成产气荚膜梭菌α-毒素基因,制成DNA标准品,选取3个稀释梯度的标准品进行重复试验,组内和组间变异系数均小于5%,证明该方法具有较好的重复性。采用7个稀释梯度的标准品进行重复检测,计算出该方法的检测限为12.39 copies/μL,定量限为33.97 copies/μL。通过对68份临床样品进行检测,证实该方法可用于临床,且用于检测质粒DNA时无明显的基质效应。应用ddPCR方法对标准品进行均匀性和稳定性检验,并联合9家实验室进行定值,最终获得了国家市场监督管理总局颁发的标准物质证书(GBW(E)091237)。α-毒素基因ddPCR方法的建立和DNA标准物的研制,为产气荚膜梭菌分子生物学检测试剂的标准化奠定了基础。
Clostridium perfringens is an important zoonotic pathogen withα-toxin gene as major molecular biological detection target.A droplet digital PCR(ddPCR)method was developed using Clper primer and probe with the optimal concentration of primer and probe 0.85 and 0.3μmol/L respectively and the annealing temperature as 60℃.The DNA of Listeria,Salmonella,Escherichia coli,Enterococcus,Campylobacter and Staphylococcus aureus were assessed by the established method,and all results were negative,indicating that the method had good specificity.Repetitive experiments based on three dilution titers of reference materials of synthesizedα-toxin DNA showed that the variation coefficients of both inter-and intra-groups were less than 5%,which indicated good repeatability.The limit of detection and the limit of quantification were determined by detecting seven dilution titers of reference materials,which were 12.39 and 33.97 copies/μL respectively.The established method was verified to be applied in clinic by detecting 68 clinical samples,and had no obvi-ous matrix effect when used for plasmid DNA detection.A reference material certificate(GBW(E)091237)had been issued for the reference material by the State Administration of Market Supervision and Administra-tion after detecting the homogeneity and stability by the ddPCR method and determining the fixed values by 9 laboratories.Overall,the establishment of the ddPCR method forα-toxin gene and the development of DNA reference materials could lay the foundation for standardization of molecular biological detection reagents for C.perfringens.
作者
张铭洋
张喜悦
赵格
曲志娜
高宏伟
孙敏
程慧敏
徐佳微
王君玮
邹明
Zhang Mingyang;Zhang Xiyue;Zhao Ge;Qu Zhina;Gao Hongwei;Sun Min;Cheng Huimin;Xu Jiawei;Wang Junwei;Zou Ming(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266000,China;China Animal Health and Epidemiology Center,Qingdao 266032,China;Qingdao Customs Technology Center,Qingdao 266000,China)
出处
《山东农业科学》
北大核心
2024年第1期156-163,共8页
Shandong Agricultural Sciences
基金
国家重点研发计划项目(2022YFD1301003,2022YFC2303900)
山东省重点研发计划项目(2022CXGC010606-01-05)
中国动物卫生与流行病学中心创新基金项目“单增李斯特菌等致病菌标准物质研究”(DW2021001-13)。
关键词
产气荚膜梭菌
ddPCR
α-毒素基因
标准物质
特异性
灵敏度
可重复性
定值
Clostridium perfringens
Droplet digital PCR
α-Toxin gene
Reference material
Specifici-ty
Sensitivity
Repeatability
Fixed value
作者简介
张铭洋(1996-),男,硕士研究生,主要从事致病微生物研究.E-mail:zhangmy19961227@163.com;第一作者:张喜悦(1974-),男,河南鹤壁人,博士,副研究员,主要从事致病微生物研究.E-mail:Zhangxiyue@cahec.cn;通信作者:邹明(1974-),男,山东龙口人,博士,教授,主要从事兽医药理与毒理学研究.E-mail:zoudnet@163.com;王君玮(1968-),男,山东临沂人,博士,研究员,主要从事致病微生物研究.E-mail:Wangjunwei@cahec.cn。
