产气荚膜梭菌α-毒素基因数字PCR方法的建立及其在标准物质研制方面的应用

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中文摘要：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。85 μmol/L，探针浓度为 0。3 μmol/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。

外文标题：Development of Digital PCR for α-Toxin Gene of Clostridium perfringens and Its Application in Preparation of Reference Materials

外文摘要：Clostridium perfringens is an important zoonotic pathogen with α-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 0.3 μmol/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 synthesized α-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 for α-toxin gene and the development of DNA reference materials could lay the foundation for standardization of molecular biological detection reagents for C.perfringens.

外文关键词：

Clostridium perfringensDroplet digital PCRα-Toxin geneReference materialSpecifici-tySensitivityRepeatabilityFixed value

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

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作者单位：

青岛农业大学动物医学院,山东青岛 266000

中国动物卫生与流行病学中心,山东青岛 266032

青岛海关技术中心,山东青岛 266000

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

基金：

国家重点研发计划项目国家重点研发计划项目山东省重点研发计划项目中国动物卫生与流行病学中心创新基金项目

项目编号：

2022YFD13010032022YFC23039002022CXGC010606-01-05DW2021001-13

出版年：

2024

DOI：

10.14083/j.issn.1001-4942.2024.01.021

山东农业科学

山东省农业科学院,山东农学会,山东农业大学

山东农业科学

CSTPCD北大核心

影响因子：0.578

ISSN：1001-4942

年,卷(期)：2024.56(1)

参考文献量26