PCRR-FLP方法在蛔虫种间鉴别上的应用  

Application of Polymerase Chain Reaction Linked Restriction Fragment Length Polymorphism in the Interspecific Differentiation of Ascarides

作　　者：蔺东[1] 刘群[1] 蒋金书[1] 

机构地区：[1]中国农业大学动物医学院,北京100094

出　　处：《畜牧兽医学报》2002年第6期585-590,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然基金资助 ( 39870 6 0 8)

摘　　要：研究应用PCR RFLP方法初步鉴别了 4种不同蛔虫。从猪蛔虫、人蛔虫、犬弓首蛔虫及鸡蛔虫中分别提取基因组DNA ,通过PCR扩增得到长度分别约为 450bp、450bp、530bp、550bp的PCR产物。经与国外报道相比较 ,确认完整扩增出 4种蛔虫的第二内部转录间隔区 (ITS 2 )片段。PCR产物经限制性内切酶HaeⅢ和HinfⅠ酶切鉴定 ,通过产生的特异性片段首先可以区分犬弓首蛔虫与鸡蛔虫。通过对 4种蛔虫的基因组DNA进行扩增 ,得到长度分别约为 980bp、980bp、1 0 50bp、1 0 70bp的PCR产物。经与国外报道相比较 ,确认完整扩增出 4种蛔虫的ITS 1、ITS 2及 5 8S核糖体DNA。PCR产物经限制性内切酶HaeⅢ酶切鉴定 ,通过产生的特异性片段可以区分出猪蛔虫与人蛔虫。通过PCR RFLP分析 ,初步推测猪蛔虫与人蛔虫仍为两个独立虫种 。We Primary distinguished the different species of Ascarides using Polymerase chain reaction linked restriction fragment length polymorphism method (PCR RFLP).Genomic DNA was extracted from Ascaris suum, A.lumbricoides,Ascaridia galli and Toxocara canis. A region spanning the second internal transcribed spacer (ITS 2) of the ribosomal DNA of each species was amplified by PCR using the primers set (0102 131,0102 132).The lengths of PCR products are 450bp,450bp,530bp and 550bp respectively.We ensured that we amplified the whole ITS 2 fragment after comparing with the previous report.PCR products were digested with two restriction endonuc lease (HaeⅢ,HinfⅠ).The two species(T.canis,A.galli) could be differentiated from other two ascaridoids through their specific bands first.A region spanning the ITS 1,ITS 2 and 5 8SrDNA was amplified from genomic DNA by PCR using the primers set(0320 144,0320 145).The lengths of PCR products are 980bp,980bp,1050bp and 1070bp respectively.We ensured that we amplified the whole ITS 1,ITS 2 and 5 8SrDNA after comparing with the previous report.PCR products were digested with Restriction endonuclease HaeⅢ.The two species(A.suum,A.lumbrico des) could be differ entiated from each other through their specific bands.We primary confered that the A.suum and A.lubricodes are still separated species through PCR RFLP analysis,but the resolution of the question requires reciprocal crossmating experiments to be done.

关 键 词：PCR-RFLP方法 蛔虫 种间鉴别 应用 聚合酶 链式反应 限制性片段长度 多态性 

分 类 号：S852.7[农业科学—基础兽医学]