副鸡嗜血杆菌16 S rDNA PCR检测方法的建立

Detection of 16 S rDNA of Haemophilus paragallinarum by PCR

根据副鸡嗜血杆菌的16 S rDNA基因序列设计一对特异性引物XZIC1和XZIC2,对6株副鸡嗜血杆菌进行PCR扩增。结果显示,该对引物对6株副鸡嗜血杆菌均扩增出与预期大小相一致的282bp片段,而对鸡毒支原体、禽巴氏杆菌、鸡传染性支气管炎病毒、鸡新城疫病毒、大肠埃希菌、鸡白痢沙门菌、禽流感病毒(H9)、鸡喉气管炎病毒及葡萄球菌等9种病原体的扩增结果均为阴性。该PCR敏感性结果表明,本方法可以检测到10pg的副鸡嗜血杆菌DNA模板。采用引物XZIC1和XZIC2,对分别用副鸡嗜血杆菌ctcc253、ctcc255、ctcc257、ctcc269株感染SPF鸡的临床病料DNA进行PCR扩增,均可扩增出单一的282bp的片段。

A transcriptase-polymerase chain reaction method was doveloped for detection of Haemophilus paragallinarum. One pair of specific primer XZIC1 and XZIC2 were designed according to the sequences of 16 S rDNA of H. paragallinarum （HPG）. The DNA of 6 HPG strains were detected by PCR using these primers. A HPG specific 282-base PCR products was amplified by these primers from these 6 HPG strains, but not from 9 other avian pathogenic viruses and bacteria such as Mycoplasma gallisepitcum, avian Pasteurlla multocida, infectious bronchitis virus, Newcastle disease virus, Escherichia coli, Salmonella pulloru, avian influenza（H9）, infectious laryngotracheitis virus and Staphylococcus. As little as 10 pg of HPG DNA was able to be detected by this method. Detection of HPG in SPF chicks artificially infected with HPG ctcc253, ctcc255, ctcc257 and ctcc269 stains by this method was achieved, and a HPG specific 282-base PCR products was amplified.