牛支原体肺炎PCR诊断方法的建立及初步应用

参照GenBank中牛支原体脂蛋白P48基因序列,设计并合成特异性引物Mb-F/Mb-R,建立了牛支原体PCR检测方法,并在扩增条件优化的基础上,对该方法的特异性和灵敏度进行了分析,进而应用建立的方法完成了疑似牛支原体肺炎临床病料的检测。结果显示,建立的PCR方法对牛支原体的核酸样品能扩增出534 bp的特异性目的片段,而对无乳支原体、丝状支原体、大肠杆菌、绿脓杆菌、链球菌、多杀性巴氏杆菌、金黄色葡萄球菌、沙门菌、肺炎克雷伯氏菌、黏质沙雷氏菌、变形杆菌等牛常见条件病原微生物核酸样品均未见明显的扩增条带,且其可检测出的牛支原体DNA最低浓度约为0.000 002 875μg/mL。对临床样品的检测结果显示,该方法的检出率与与病原分离的符合率为100%。上述结果表明,本研究成功建立了牛支原体PCR检测方法,可用于临床上牛支原体肺炎的快速诊断。

Establishment and preliminary application of PCR diagnostic method for Mycoplasma bovis pneumonia

According to the sequence of lipoprotein P48 gene of Mycoplasma bovis(Mb) in GenBank,the specific primer pairs Mb-F/Mb-R were designed and synthesized.And the PCR method was established for detection of Mb.Then based on the optimization of PCR detection conditions,the specificity and sensitivity of PCR were analyzed.Subsequently,the clinical samples collected from the calf with suspected case of Mb pneumonia in different regions in Gansu province were detected using the developed method.The results showed that a 534 bp DNA fragment was amplified from the DNA of Mb using the established PCR method,whereas no amplification was found for Mycoplasma agalactiae,Mycoplasma mycoides subsp,mycoides SC(MmmSC),Escherichia coli,Pseudomonas aeruginosa,Streptococcus,Pasteurella multocida,Staphylococcus aureus,Salmonella,Klebsiella pneumoniae,Serratia marcescens,Serratia symbiotic,Proteus species,etc.The minimum detectable concentrations of the PCR for the DNA of Mb was 0.000 002 875 μg/mL.And the detection rate of clinical samples with the established PCR was 100% conform with those of the Mb culture test.This study successfully established a PCR assay for detecting Mb,which can be applied to the rapid diagnosis of Mb pneumonia in clinic.