绵羊支原体肺炎病原巢式PCR检测方法的建立与应用

Establishment and Application of Nested PCR Detection Method of Pathogens of Mycoplasma ovipneumonia

[目的]建立一种诊断绵羊支原体肺炎病原的巢式PCR方法,确定肺炎支原体感染的靶器官。[方法]根据GenBank网站上登录的绵羊支原体16S rRNA基因序列,设计并合成2对引物,以肺炎支原体菌株基因组DNA为模板,经过PCR反应条件的优化,通过测序验证扩增产物的正确性,建立了绵羊支原体肺炎病原的巢式PCR检测方法,进而应用建立的方法完成临床阳性病料肺脏、肺淋巴、心脏、肾脏、肝脏、脾脏、皮肤、小肠和外周血检测以及疑似样本肺脏组织的检测。[结果]建立的巢式PCR方法可扩增出864 bp的特异性目的片段,肺脏和肺淋巴为绵羊肺炎支原体感染的靶器官,临床样本巢式PCR检出率与支原体培养鉴定结果的符合率为100%。[结论]建立的绵羊支原体肺炎病原巢式PCR检测方法可用于临床样本的实验室诊断。

[Objective]In order to establish a nested polymerase chain reaction(PCR)for detecting the pathogens of Mycoplasma ovipneumonia,and determine the target organs of mycoplasma infection.[Method]According to the 16S rRNA sequence of sheep mycoplasma in GenBank website,two pairs of specific primers were designed and synthesized.PCR reaction conditions were optimized by using the genome DNA of M.ovipneumonia as templates.The nested PCR products were verified by sequencing.A nested PCR method for detecting the pathogens of M.ovipneumonia was established.The established method was used to detect the lung,lung lymph,heart,kidney,liver,spleen,skin,small intestine and peripheral blood of clinical positive samples,and the lung tissue from clinical suspected samples.[Result]864 bp specific gene fragment was amplified by using nested PCR method.The lung and lung lymph of sheep were the target organs of mycoplasma infection.The coincidence rate between the detection rate of clinical samples by the nested PCR and pathogen culture and identification results was 100%.[Conclusion]The nested PCR method for detection of M.ovipneumonia could be applied for the laboratory diagnosis of clinical samples.