兔肠道球虫病三重PCR方法的建立与应用  

Establishment and application of triplex PCR method for rabbit intestinal coccidiosis

作　　者：闵德省 包涛涛 鲜思美[1,4] 顾庆林 梁倩 杨倩 郑维豪 MIN Desheng;BAO Taotao;XIAN Simei;GU Qinglin;LIANG Qian;YANG Qian;ZHENG Weihao(College of Animal Science,Guizhou University,Guiyang 550025,China;Pu’an County Secondary Vocational School,Pu’an 561500,China;Guizhou Province Qiandongnan Prefecture Bureau of Agriculture and Rural Affairs,Qiandongnan 556000,China;Guizhou Province Institute of Animal Disease Research,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]普安县中等职业学校,贵州普安561500 [3]贵州省黔东南州农业农村局,贵州黔东南556000 [4]贵州省动物疫病研究所,贵阳550025

出　　处：《黑龙江畜牧兽医》2023年第20期72-76,82,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：贵州大学博士基金项目(贵大人基合字[2020]69号)。

摘　　要：为建立一种能同时检测兔穿孔艾美耳球虫、黄艾美耳球虫、大型艾美耳球虫的三重PCR方法,试验首先根据GenBank中的兔穿孔艾美耳球虫Eper基因、黄艾美耳球虫Efla基因、大型艾美耳球虫Eman基因序列设计3对特异性引物,通过优化PCR反应的退火温度和引物浓度,建立一种用于检测兔肠道球虫病的三重PCR方法,然后对该方法的特异性、敏感性及重复性进行评价,最后分别采用该方法及单一PCR方法对82份临床样本进行检测,计算两种方法的符合率。结果表明:所建立的三重PCR方法的最佳退火温度为59.7℃,最佳引物(F/R)浓度为0.15/0.15,0.20/0.20,0.20/0.20μmol/L(Eper、Efla、Eman基因);该方法能特异性扩增出兔穿孔艾美耳球虫、黄艾美耳球虫、大型艾美耳球虫三种兔球虫卵囊靶基因,而斯氏艾美耳球虫、大肠杆菌及沙门氏菌的检测结果为阴性;能够检测出穿孔艾美耳球虫、黄艾美耳球虫、大型艾美耳球虫三种兔球虫卵囊基因组DNA的最低限值分别为6.0,6.4,8.0 pg,两种模板浓度组合(60,64,80 ng/μL和6.0,6.4,8.0 ng/μL)批内重复性试验和批间重复性试验所有重复均扩增出预期目的条带;临床样本中,该方法的单一球虫检出率为53.6%,两种球虫检出率为30.5%,三种球虫检出率为7.3%;除黄艾美耳球虫+大型艾美耳球虫的混合感染类型两种方法检测结果的符合率为91.8%外,其他感染类型两种方法检测结果的符合率均为100%,整体符合率为98.8%。说明成功建立了一种可用于检测兔穿孔艾美耳球虫、黄艾美耳球虫、大型艾美耳球虫的多重PCR方法,该方法具有特异性好、灵敏度高、重复性好的特点。In order to establish a triplex PCR method that could simultaneously detect Eimeria perforans,Eimeria flavescens and Eimeria maxima,in this experiment,firstly,three pairs of specific primers were designed according to the sequences of Eper gene of rabbit Eimeria perforans,Efla gene of Eimeria flavescens and Eman gene of Eimeria maxima.By optimizing the annealing temperature and primer concentration of the PCR reaction,a triplex PCR method for the detection of rabbit intestinal coccidiosis was established.Then,the specificity,sensitivity and reproducibility of the method were evaluated,and finally and a single PCR method the triplerx PCR method were used to detect 82 clinical samples;the compliance rate of the two methods was calculated.The results showed that the optimal annealing temperature of the established triplex PCR method was 59.7℃.The optimal primer(F/R)concentrations were 0.15/0.15,0.20/0.20,0.20/0.20μmol/L(Eper,Efla and Eman genes).This method could specifically amplify the target genes of oocysts of three rabbit coccidia,Eimeria perforans,Eimeria flavescens and Eimeria maxima;while the test results of Eimeria stiedai,Escherichia coli and Salmonella were negative.The minimum detection limits for three kinds of oocyst genomic DNA of Eimeria perforans,Eimeria flavescens and Eimeria maxima were 6.0,6.4 and 8.0 pg,respectively.All replicates of two kinds of template concentration combination(60,64,80 ng/μL and 6.0,6.4,8.0 ng/μL)of intra-batch and inter-batch repeatability tests amplified the prospected target bands.In clinical samples,the detection rate of single coccidia was 53.6%;that of two coccidia was 30.5%;that of three coccidia was 7.3%.Except for the mixed infection type of Eimeria flavescens+Eimeria maxima with the consistency rate of the test results of the two methods being 91.8%,the consistency rates of the test results of the other infection types were 100%,the overall coincidency rate was 98.8%.The results suggested that a multiplex PCR method for the detection of rabbit Eimeria perforan

关 键 词：兔球虫 穿孔艾美耳球虫 黄艾美耳球虫 大型艾美耳球虫 三重PCR方法 

分 类 号：S855[农业科学—临床兽医学]