摘要
根据GenBank中16株鸭疫里默氏杆菌(RA)的外膜蛋白A(OmpA)基因序列,在其保守区设计了一对特异性引物,建立了PCR方法,并对已有5个血清型的鸭疫里默氏杆菌纯培养菌和6株临床分离未定型的里默氏杆菌菌株,及病死鸭病料组织进行了检测。结果表明,5个血清型的鸭疫里默氏杆菌纯培养菌和6个未定型的里默氏杆菌的DNA都可扩增出592 bp的特异目的条带,而对鸭源大肠杆菌、鸭源多杀性巴氏杆菌、鸭源沙门氏菌的扩增结果均为阴性,在对11只不同鸭场病死鸭各种组织的检测中,脑的检出率为5/11(高于细菌分离的3/11),肝脏的检出率为4/11(高于细菌分离的3/11),心血的分离率为3/11(等于细菌分离的3/11),其它脏器未检测到里默氏杆菌。分别将鸭疫里默氏菌基因组DNA和菌落提取液进行10倍梯度稀释,基因组DNA的最小检出量为10 pg,菌落最小检出量为10 CFU/m l。此法可用于快速鉴定鸭疫里默氏杆菌,也适用于病料的直接检测。
According to the gene sequences of the outer membrane protein A （OmpA） from 16 Riemerella anatipestifer （RA） strains in the GenBank, a pair of specific primers was designed in those conservative regions. Then a PCR technique was established to detect 5 serotype and 6 unknown type RA strains which grew on TSA medium and samples from different duck farms. The results showed that a 592 bp DNA fragment was amplified as it was expected from all the detected RA strains, but Escherichia coli, Pasteurella multocida, Salmonella isolated from duck revealed negative results. Of 11 ducks from different farms, 5,4 and 3 ducks gave positive results from brain and liver tissues and heart blood respectively by PCR, while those were 3 ducks by bacteria isolation. The RA genomic DNA and colony extraction were gradient diluted for 10 times to test the accuracy and sensitivity of this PCR method. As a result, the detection limit of this method was 10 pg genomic DNA or 10 CFU/ml colony. So this PCR assay provided a rapid and accurate method for identification of RA.
出处
《山东农业科学》
2009年第8期107-110,共4页
Shandong Agricultural Sciences
