鸡毒支原体实时荧光定量PCR检测方法的建立

Development of Real-Time Fluorescent Quantitative PCR Method for Detection of Mycoplasma gallisepticum

【目的】建立基于鸡毒支原体种特异性粘附蛋白编码基因pvpA的实时荧光定量PCR检测方法。【方法】根据GenBank公布的不同国家和地区鸡毒支原体pvpA基因序列,在高度保守区域设计一对引物。以临床分离鸡毒支原体RC1为模板扩增pvpA基因,将其连接到PMD19-T载体,转化至大肠杆菌DH5α中,经PCR和酶切鉴定并测序验证后得到阳性重组质粒rPvpA90。以rPvpA90为模板建立SYBR Green I荧光定量的标准曲线和溶点曲线,并进行特异性,灵敏性,重复性及临床样本检测试验,评价该方法的可行性。【结果】所建立的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶点曲线特异,相关系数为0.990。最低检测限为72拷贝/20μL,其敏感性比常规PCR至少高100倍;无论是对不同病原DNA单模板还是几种病原DNA混合模板进行扩增,该方法都呈现很好的特异性;重复性试验中,批内和批间变异系数均小于2%,表明该方法重现性好;临床样本的检测结果表明所建立的荧光定量PCR检测方法的检测率明显高于常规PCR方法。【结论】本研究初步建立了基于种特异性基因pvpA的鸡毒支原体荧光定量PCR方法,为养禽场诊断和监测鸡毒支原体病原提供一种新的特异、灵敏的方法。

【Objective】The objective of this study is to develop a SYBR Green I Real-Time PCR assay with species-specific surface-exposed protein gene pvpA as the target for detection of Mycoplasma gallisepticum.【Method】A pair of primers was designed within highly conservative region of M.gallisepticum pvpA gene following BLASTn all published pvpA sequences accessible to GenBank.Ninety bp fragment of pvpA was amplified from M.gallisepticum isolate,then cloned into PMD19-T vector.Confirmed with PCR or digestion with EcoRⅠand Hind Ⅲ,a recombinant plasmid rPvpA90 was obtained when competent E.coli DH5α was transformed with the PMD19-pvpA vector.The positive plasmid rPvpA90 was used as the quantitative template to generate standard curve and melt curve.Analytical specificity,sensitivity,and reproducibility were evaluated respectively and clinical sampiles were detected.【Result】 The results demonstrated standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melt curve was specific with the correlation coefficient of 0.990.The test had a detection limit of 74 copies per 20 μL when tested with M.gallisepticum genomic DNA.The sensitivity of the assay was at least 100-fold higher than that of the conventional PCR.The assay was confirmed to be highly specific when amplified with either single or mixed DNA sample from microorganisms.The variation coefficient of Ct values of diluted standard DNA was less than 2%,which indicated a good reproducibility.The test result of clinical samples demonstrated that the detection rate of the assay was significantly higher than that of the conventional PCR.【Conclusion】The developed Real-Time PCR assay was highly specific,sensitive,and reproducible and could be a potential tool for diagnosis and monitoring of M.gallisepticum in poultry farm.