梨形虫和伊氏锥虫双重PCR检测方法的建立  

Establishment of a duplex PCR detection method for Piroplasmida and Trypanosoma evansi

作　　者：孔令丽 李祥龙 章志涛 罗雨昕 王冬英[1,2] KONG Lingli;LI Xianglong;ZHANG Zhitao;LUO Yuxin;WANG Dongying(College of Animal Scienceand Technology,Guangxi University,Nanning 530004,China;Guangxi Zhuang Autonomous Region Engineering Research Center for Veterinary Biologics,Nanning 530004,China)

机构地区：[1]广西大学动物科学技术学院,南宁530004 [2]广西壮族自治区兽用生物制品工程研究中心,南宁530004

出　　处：《黑龙江畜牧兽医》2023年第4期72-76,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：崇左市科技计划项目(崇科FA2019006);国家寄生虫资源中心项目(NPRC-2019-194-30);广西创新驱动发展专项(桂科AA17204057)。

摘　　要：为了建立梨形虫(Piroplasmida)和伊氏锥虫(Trypanosoma evansi)的双重PCR检测方法,试验根据梨形虫的18S rRNA基因和伊氏锥虫的kDNA基因设计两对特异性引物,以吕氏泰勒虫和伊氏锥虫阳性样品DNA为模板进行单重PCR和双重PCR,并对双重PCR的退火温度进行优化,对本研究建立并优化的双重PCR方法进行特异性试验、敏感性试验及临床样品的检测。结果表明:本研究建立的双重PCR方法能够扩增出350 bp和600 bp的特异性目的条带,而无浆体、肝簇虫、弓形虫和埃立克体样品均呈阴性,梨形虫和伊氏锥虫最低检测浓度分别为54.70 fg/μL和14.30 fg/μL,用本试验建立的方法对临床样品的检测结果与对照方法(巢式PCR)检测结果一致。说明本研究成功建立了灵敏、快速、简便、特异性高的双重PCR检测方法,能够用于梨形虫和伊氏锥虫的临床检测及流行病学调查。In order to establish a duplex PCR detection method for Piroplasmida and Trypanosoma evansi,the experiment was conducted to design two pairs of specific primers based on the 18S rRNA gene of Piroplasmida and the kDNA gene of Trypanosoma evansi;single PCR and deplex PCR were carried out by using the positive sample DNA containing Theileria luwenshuni and Trypanosoma evansi as templates,and the annealing temperature of deplex PCR was optimized.Specificity test,sensitivity test and clinical sample detection were performed by the duplex PCR method established and optimized in this study.The results showed that the duplex PCR method established in this study was able to amplify specific target bands of 350 bp and 600 bp.While Anaplasma,Hepatozoon,Toxoplasma gondii and Ehrlichia samples were negative and the lowest detected concentrations were 54.70 fg/μL and 14.30 fg/μL for Piroplasmida and Trypanosoma evansi,respectively.The results of the clinical samples tested by the method established in this test were consistent with those of the control method(nested PCR).The results suggested that this study successfully established a sensitive,rapid,simple and highly specific duplex PCR detection method that could be used for the clincal detection of Piroplasmida and Trypanosoma evansi,and epidemiological investigation.

关 键 词：梨形虫 伊氏锥虫 PCR 检测方法 应用 

分 类 号：S852.7[农业科学—基础兽医学] Q781[农业科学—兽医学]