3种致毛皮动物肺炎病原菌多重PCR检测方法的建立

Establishment of multiplex PCR method for dectection of three pathogenic bacteria causing the pneumonia of fur-bearing animal

大肠杆菌、铜绿假单胞菌和肺炎克雷伯菌是引起毛皮动物肺炎的最为常见的细菌性病原。本研究选择3种细菌的特异性基因,分别为phoA(大肠杆菌)、plcH(铜绿假单胞菌)和khe(克雷伯菌)基因,进行多重PCR方法的建立,预期扩增目的条带大小分别为720,199,428bp。通过反应条件的优化,选择退火温度54.5℃,引物体积(按照phoA、plcH和khe顺序,浓度10μmol/L)为0.50,0.75,2.00μL。敏感性试验结果显示,大肠杆菌和肺炎克雷伯菌最低检测DNA质量浓度为1mg/L (1μL),而铜绿假单胞菌的最低检测质量浓度为100μg/L (1μL)。利用建立的多重PCR方法对临床样品进行检测,结果表明该方法能够有效检测出3种病原菌。

Escherichia coli,Pseudomonas aeruginosa and Klebsiella pneumonia are the most common pathogenic bacteria that cause the pneumonia of fur-bearing animal.In this study,three specific genes,such as phoA gene(Escherichia coli),plcH gene(Pseudomonas aeruginosa)and khe gene(Klebsiella pneumonia),were selected to establish the multiplex PCR method,and the predicted size were 622 bp,199 bp and 428 bp,respectively.Under the optimized reaction conditions,54.5℃ was selected as the best annealing temperature and 0.50μL(phoA),0.75μL(plcH)and 2.00μL(khe)was the best primers volume(10μmol/L).The sensitivity test showed that the multiplex PCR could detect the genome DNA of 1 mg/L(1μL)for Escherichia coli and Klebsiella pneumonia,and 100μg/L(1μL)for Pseudomonas aeruginosa.Clinical sample detection showed that the multiplex PCR could effectively detect the three kinds of bacteria.