Establishment of a fluorescence quantitative PCR detection method for Strepto-coccus equi subspecies zooepidemicus and its application in retrospective detection of imported horses
Establishment of a fluorescence quantitative PCR detection method for Strepto-coccus equi subspecies zooepidemicus and its application in retrospective detection of imported horses
胡雨桐 1周学慧 1赵梦茹 1陈翔 2吴晓薇 3赵治国 4王艳 2赵光伟1
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作者信息
1. 西南大学动物医学院,重庆荣昌 402460
2. 上海海关,上海 200135
3. 广州海关技术中心,广东广州 510623
4. 呼和浩特海关技术中心,内蒙古呼和浩特 010010
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摘要
为建立一种针对马链球菌兽疫亚种(Streptococcus equi subspecies zooepidemicus,SEZ)快速、特异、敏感的检测方法,并了解SEZ在入境我国马匹中的感染情况,试验首先根据SEZ标准菌株(ATCC 43079)的保守基因comB设计合成特异性引物,经PCR扩增后构建pMD19-T-comB重组质粒,以此为标准品模板建立基于SYBR Green Ⅰ染料的荧光定量PCR检测方法(quantitative PCR,qPCR);进而利用所建方法对2018-2023年由荷兰、比利时、日本、德国、阿根廷和新西兰等6个国家入境我国口岸的477份马血清样本进行SEZ感染情况的检测分析.结果发现本试验所建立的qPCR检测方法特异性良好,仅对SEZ存在特异性扩增;敏感性试验表明该方法最低检测量为4.58 × 101 copies/μL;重复性试验检测结果显示批内重复性变异系数均小于0.5％,批间重复性变异系数均低于3.0％,表明重复性良好,稳定性高.回顾性检测分析显示2018年荷兰入境的共40份样本均为阴性(0/40);2019年比利时入境样本中检测到1份阳性(1/20),日本和德国入境样本均为阴性(0/36);2021年日本样本中3份为阳性(3/34),阿根廷1份为阳性(1/20),荷兰均为阴性(0/40);2022年荷兰76份样本均为阴性(0/76);2023年荷兰126份样本检出阳性样本5份(5/126),新西兰88份样本中检出阳性1份(1/88).总计阳性样本11份,阳性率为2.31％(11/477),总体阳性率较低.结果说明,成功建立了用于检测SEZ的SYBR Green Ⅰ荧光定量PCR检测方法,其对我国出入境、口岸部门的快速检疫以及该病的流行病学调查等工作提供了必要的技术支撑.
Abstract
In order to establish a rapid,specific and sensitive detection method for Streptococcus equi subspecies zooepidemicus(SEZ)and to understand the infection status of SEZ in horses ente-ring China,specific primers were designed and synthesized based on the conserved gene comB of standard strain SEZ(ATCC 43079)in this work.Then,the pMD19-T-comB recombinant plasmid was constructed and used as a standard positive template.After that,the fluorescence-based quantitative PCR(qPCR)detection method based on SYBR Green Ⅰ dye was established.Totally,477 equine entry serum samples from 6 countries,including Netherlands,Belgium,Japan,Germa-ny,Argentina and New Zealand,during 2018 to 2023,were randomly selected and detected for SEZ by the qPCR method.Results showed that the established qPCR method had specific amplification for only SEZ,which illustrated a good specificity.Sensitivity test of the method showed that the limited detection amount was 4.58 X101 copies/μL.And the repeatability test showed that the coef-ficient of variation of intra-batch repeatability was less than 0.5％,while the inter-batch repeat-ability was less than 3.0％,which indicated good repeatability and high stability.Retrospective a-nalysis showed that totally 11 of 477 positive samples were detected,with a relatively low positive rate of 2.31％(11/477).Among them,all the 40 samples from Netherlands in 2018 were negative(0/40).In the samples of 2019,one positive was detected from Belgium(1/20),while all other 36 samples which form Japan and Germany were negative.In the samples of 2021,three samples(3/34)from Japan and one sample(1/20)from Argentina were positive,and all the other 40 samples from the Netherlands were negative.In the samples of 2022,76 samples from Netherlands were all negative.While in the 2023,5(5/126)of 126 samples from Netherlands and one(1/88)of 88 from New Zealand were found positive with SEZ.To summarize,The SYBR Green Ⅰ qPCR method for the diagnosis of SEZ was successfully established,and it could provide necessary technical support for the rapid quarantine of China's entry-exit and port departments,as well as the epidemiological investigation of the disease.
关键词
马链球菌兽疫亚种/荧光定量PCR/SYBR/Green/Ⅰ/马
Key words
Streptococcus equi subspecies zooepidemicus/fluorescence quantitative PCR/SYBR Green Ⅰ/horse
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