四种家兔呼吸道疾病病原多重PCR方法的建立与应用

Establishment and application of a multiplex PCR for simultaneous detection of four pathogens resulted in respiratory syndrome in rabbit

为建立同时检测家兔肺炎克雷伯菌(K.pneum oniae)、兔病毒性出血症病毒(RH D V)、多杀性巴氏杆菌(P.m ultocida)和支气管败血波氏杆菌(B.bronchiseptica)的多重PC R方法,根据G en Bank中肺炎克雷伯菌的16S r RN A基因、RHDV的VP60基因、多杀性巴氏杆菌的ktm1基因和支气管败血波氏杆菌的ptx A基因的保守序列设计4对特异性引物,优化反应体系与反应条件,建立能同时扩增兔肺炎克雷伯菌(127bp)、RHDV(322 bp)、多杀性巴氏杆菌(457 bp)和支气管败血波氏杆菌(872 bp)相应基因的四重PCR方法。结果显示,该方法能单管同时特异性检测兔源肺炎克雷伯菌、RHDV、多杀性巴氏杆菌与支气管败血波氏杆菌,且对兔源金黄色葡萄球菌、无乳链球菌、大肠杆菌与魏氏梭菌等无特异性扩增,说明该方法具有较强的特异性。该方法的灵敏度分别为9.16、7.24×10-3、7.96、6.8×10-1pg/L。采用建立的方法对人工感染试验病例与临床样本检测结果表明,该方法能快速、特异地鉴别诊断4种病原的单独与混合感染,与单一PCR方法相比,无显著性差异(P>0.05)。该方法的建立为临床上鉴别诊断4种家兔呼吸道疾病病原提供了重要的技术手段。

To establish a multiplex PCR for simultaneous detection of Klebsiella pneumoniae,rabbit haemorrhagic disease virus（RHDV）,Pasteurella multocida and Brodetella bronchiseptica,four pairs of specific primers were designed according to the conservative regions of 16 S r RNA gene for K.pneumoniae,VP60 gene for RHDV,ktm1 gene for P.multocida and ptx A gene for B.bronchiseptica,then the reaction system and conditions were optimized to establish the quadruple PCR for the detection on K.pneumoniae（127 bp）,RHDV（322 bp）,P.multocida（457 bp） and B.bronchiseptica（872 bp）.In result,the method can identify K.pneumoniae,RHDV,P.multocida and B.bronchiseptica specifically and simultaneously from the same sample containing the four pathogens,and non-specifically amplify for S.aureus,S.agalactiae,E.coli and C.welchii.Anditsdetectionsensitivityforfourpathogensare9.16pg/ L（K.pneumoniae）,7.24×10-3pg/ L（RHDV）,7.96 pg/ L（P.multocida） and 6.8×10-1pg/ L（B.bronchiseptica）,respectively.The results of the detection on the samples of the four pathogens artificial infection and clinical showed that the developed multiplex PCR could be uesed to diagnose the four pathogen samples or mixed infection specifically and quickly.While,there is no significant difference compared to the single PCR.So the established method can provide a powerful technical means simultaneous diagnosis of the four pathogens in clinical.