兔斯氏艾美耳球虫荧光定量PCR检测方法的建立

摘要

Objective To develop the method for quantitatively detecting Eimeria stiedai (E.stiedai) by Real-Time PCR (RT-PCR).Methods Specific primers were designed and synthesized according to a part of the sequence of ITS1 region ofE.stiedai.The recombinant plasmid was constructed to established the standard curve of RT-PCR.The specificity,sensitivity and reproducibility of the RT-PCR method were performed.Result The method was specific and sensitive for detection ofE.stiedai.Meanwhile,the sensitivity of the present method could detect the DNA of one E.stiedai and the variation coefficients of intra-assay and inter-assay were less than 2.0％ respectively.Conclusion The established RT-PCR is a specific,sensitive and reliable method for the quantitative detection ofE.stiedai.%目的 建立荧光定量PCR检测兔斯氏艾美耳球虫的方法.方法 根据斯氏艾美耳球虫(内转录间隔区1)ITS1序列区设计特异性引物,构建重组质粒,并作为标准品绘制标准曲线,对建立的荧光定量PCR检测方法进行特异性、敏感性和重复性试验.结果 建立的荧光定量PCR能特异性地检测兔斯氏艾美耳球虫,且可检测到含一个卵囊DNA的样本.该方法的重复性较好,组内、组间重复试验的变异系数均小于2％.结论 建立了一种特异性好、敏感度高、可靠的检测兔斯氏艾美耳球虫荧光定量PCR方法.