鸡毒支原体鸡滑液囊支原体和副鸡禽杆菌三重PCR检测方法的建立

鸡毒支原体(MG)、鸡滑液囊支原体(MS)、副鸡禽杆菌(APG)三重PCR诊断对于鸡呼吸道疾病的鉴别诊断和防控有着重要意义。本次试验首先对三重PCR反应条件进行优化,之后对多种病原的DNA进行扩增以确定本方法的特异性,调整MG、MS和APG的DNA浓度以确定本方法的敏感性。结果显示,利用优化后的反应条件,本试验建立的诊断方法能够同时扩增出长度为453 bp(MG)、328 bp(MS)和241 bp(APG)的特异性片段,而大肠杆菌、鸡白痢沙门菌、禽多杀性巴氏杆菌、金黄色葡萄球菌和阴性对照样品在检测时则无条带产生。对三种病原的混合DNA进行10倍梯度稀释,确定MG、MS和APG的最低检出量为5×10^-3ng/μL。对37份临床样品的检测结果表明,三重PCR的检出率和单重PCR的检测结果一致,且可同时检测出两种或三种病原同时感染。上述结果表明,本试验建立的MG、MS和APG三重PCR诊断方法具有良好的特异性、较高的灵敏性,可用于鸡呼吸道病的诊断和防控。

Establishment of a triple PCR for the detection of Mycoplasma gallisepticum,Mycoplasma synoviae and Avibacterium paragallinarum

The triple PCR diagnosis of Mycoplasma gallisepticum(MG),Mycoplasma synoviae(MS)and Avibacterium paragallinarum(APG)is of great significance for the differential diagnosis and prevention and control of respiratory diseases in chickens.In this experiment,the reaction conditions of triple PCR were optimized,and then the DNA of multiple pathogens were amplified to determine the specificity of this method,and the DNA concentrations of MG,MS and APG were adjusted to determine the sensitivity of this method.The results showed that using the optimized reaction conditions,the diagnostic method established in this experiment could amplify the specific fragments with lengths of 453 bp(MG),328 bp(MS)and 241 bp(APG)at the same time.There were no bands in Escherichia coli,Salmonella pullorum,avian Pasteurella multocida,Staphylococcus aureus and negative control samples.The mixed DNA of the three pathogens were diluted by 10 fold dilution,and the minimum detection amount of MG,MS and APG was 5×10^-3 ng/μL.The detection results of 37 clinical samples showed that the detection rate of triple PCR was consistent with that of single PCR,and two or three pathogens could be detected at the same time.The above results show that the triple PCR diagnostic method of MG,MS and APG established in this experiment has good specificity and high sensitivity,and can be used in the diagnosis,prevention and control of respiratory diseases in chickens.