基于鸭疫里默氏杆菌16SrRNA PCR检测方法的建立和应用

根据GenBank中鸭疫里默氏杆菌(RA)16SrRNA基因序列设计特异性引物,建立了基于鸭疫里默氏杆菌16SrRNA PCR检测方法。以建立的PCR方法对RA、大肠杆菌、沙门氏菌、鸭源金黄色葡萄球菌、两双歧杆菌、短乳酸杆菌、屎肠球菌和短小芽孢杆菌进行检测,结果只有RA DNA能扩增出844 bp的特异条带,其他细菌的DNA不能扩增出任何条带;从凝胶中回收DNA条带,测序结果与GenBank中RA相应序列完全一致。对不同稀释度的RADNA样品进行扩增,PCR对RA DNA最小检出量为22 pg,最少检出RA 80 CFU/mL。以该PCR方法对可疑为RA的临床病料和菌株进行检测和鉴定,结果与细菌分离鉴定吻合率为100%。表明本研究所建立的基于RA 16SrRNAPCR方法具有快速、灵敏、特异的优点,可用于RA感染的检测、分子流行病学调查和分离菌株的快速鉴定。

Development and Application of PCR Based on 16SrRNA for the Detection of Riemerella anatipestifer

Primers are designed based on the conserved region of the 16SrRNA nucleotide sequences of Riemerella anatipestifer(RA) in GenBank,and the specific PCR assay for RA detection is developed.Using this assay,we can amplify a differential fragment about 844 bp from RA,but we cannot get the same fragment from Escherichia coli,Salmonella,Staphylococcus aureus,Bifidobucterium pullorum,Lactobacillus aviarius,Enterococcus faecium and Bacillus subtilis.The DNA band was obtained from agarose gel,and the sequence of the PCR product is aligned with the available sequence in GenBank.A series of sensitivity experiments is performed and proves that the detection limit of this assay is 22 pg genomic DNA and 80 CFU/mL.The clinical samples and bacteria isolated from the infected ducks are detected by the PCR assay and the positive coincidence rate comparing RA isolation with PCR is 100 percent.The result shows that the PCR assay is rapid,sensitive and specific which provides a greatly improved tool for diagnosing the infected ducks,doing molecular epidemiology survey of RA and quickly detecting the isolated bacteria.