摘要
为了建立鸡球虫种类的快速分子生物学鉴定方法,检测实验室保存虫株受污染状况,分别对单卵囊分离繁殖及实验室长期传代保存的6株巨型艾美耳球虫（EMSH01、EM4101、EMES01、EMBA01、EMTY01和EMTO01）、4株柔嫩艾美耳球虫（ETDS01、ETGD01、ETAD和ETAM）、1株堆形艾美耳球虫（EA1201）、1株变位艾美耳球虫（EMIS01）、1株毒害艾美耳球虫（ENGD01）收集卵囊,纯化、提取总DNA,根据巨型艾美耳球虫、柔嫩艾美耳球虫、堆形艾美耳球虫的RAPD和SCAR分子标记、ITS-1区序列分别设计Tn-F与Tn-R、Mx-F与Mx-R、Ac-F与Ac-R、ET-1与ET-2、EM-1与EM-2、EA-1与EA-2等6对特异性引物,对13个虫株分别进行PCR扩增,1%琼脂糖电泳分析片段大小.结果显示：用引物Tn-F与Tn-R、ET-1与ET-2对ETDS01、ETGD01、ETAD、ETAM、EMTO01扩增出特异性条带,其余4种8个虫株未见条带;用引物Mx-F与Mx-R、EM-1与EM-2对EMSH01、EM4101、EMES01、EMBA01、EMTY01和EMTO01扩增出特异性条带,其余4种7个虫株未见条带;用引物Ac-F与Ac-R、EA-1与EA-2对EA1201扩增出特异性条带,其余4种12个虫株未见条带.结果说明特异性引物PCR方法鉴定的5种球虫与原常规生物学方法鉴定的结果一致,检测出保存的巨型艾美耳球虫EMTO01虫株受柔嫩艾美耳球虫污染,其余虫株未发现交叉污染.研究证明利用特异性引物建立的PCR方法可用于鸡球虫种类与卵囊纯度的鉴定.
In order to establish a rapid molecular method for identifying different species of Eimeria and test oocysts purity of the strains of Eimeria preserved in laboratory, some strains from the single oocyst isolation and laboratory long-term preserved laboratory strains were propagated. These strains included six strains of E. maxima （ EMSH01, EM4101, EMES01, EMBA01, EMTY01 and EMTO01 ）, four strains of E. tenella （ETDSO1, ETGD01, ETAD and ETAM）, one strain of E. acervulina （ EA1201） , one strain of E. mivati （ EMISO1） and one strain of E. necatrix （ ENGD01） . DNAs were extracted from thirteen strains of Eimeria and amplified by PCR. Six pairs of specific primers in the PCR amplification were designed according to the RAPD and SCAR molecular markers and ITS-1 sequences of E. maxima, E. tenella and E. acervulina. They were Tn-F and Tn-R, Mx-F and Mx-R, Ac-F and At-R, ET-1 and ET-2, EM-1 and EM-2, EA-1 and EA-2. PCR products were assessed on 1% agarose gel electrophoresis. The results showed that specific PCR products were amplified of Eimeria by primers Tn-F and Tn-R, ET-1 and ET-2 from five strains, including ETDS01, ETGD01, ETA13, ETAM, EMT001, but not from other eight strains of four species. Another of speeific PCR produets were amplified by primers Mx-F and Mx-R, EM-1 and EM-2 from six strains of Eimeria, including EMSH01, EM4101, EMES01, EMBA01, EMTY01 and EMTO01. The third specific PCR products were amplified by Ac-F and Ac-R, EA-1 and EA-2 from only one strain EA1201. PCR results were coincidence with those general biology, only one strain of E. maxima （EMTO01） was polluted by E. tenella and other strains were not polluted. The study demonstrated that PCR method can be used to identify different Eimeria spp. and oocysts purity by genus-specific primers.
出处
《寄生虫与医学昆虫学报》
CAS
2010年第4期203-207,共5页
Acta Parasitologica et Medica Entomologica Sinica
基金
国家科技基础条件平台建设项目（2005DKA21104）
