猪附红细胞体特异性PCR检测方法的建立

从猪血中分离附红细胞体,提取其基因组DNA,用EcoRI对基因组DNA进行酶切,将酶切片段进行琼脂糖电泳,得到一个1.8 kb的DNA片段,根据DNA序列分析与GenBank已知基因比较和PCR方法确定该片段具有特异性,以该片段为模板进行PCR,并将其扩增产物与其它猪的其他细菌DNA扩增产物相比较,建立一种检测猪外周血中附红细胞体的特异性PCR检测方法,并通过斑点杂交试验检测该产物特异性,通过临床应用检验该法的适用性。结果表明:扩增产物大小为782 bp,其序列与已知附红细胞体序列同源性为100%,斑点杂交试验证实该产物为附红细胞体特异性产物,将该方法应用于检测10头临床症状典型的感染猪和10头健康猪的血液,所有感染和隐性带菌猪的PCR检测结果为阳性,健康猪为阴性。猪外周血中附红细胞体的特异性PCR检测方法适用于检测无临床症状的隐性带菌动物。

A specific polymerase chain reaction assay for detection of Eperythrozoon suis in pigs

To develop a polymerase chain reaction(PCR)assay for the identification of Eperythrozoon suis infected and latent-stage pigs,a 1.8 kb EcoRI DNA fragment of the E.suis genome was isolated from the blood of pigs experimentally infected with E.suis Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment.The homologies of nucleotide sequence of EcoRI DNA fragment of the Eperythrozoon suis Xin Xiang strains with other strains on GenBank were 100%.PCR products were 782 bp in size and were amplified only from E.suis particles prepared from the blood of experimentally infected pigs but not from any controls,comprising blood from healthy piglets and a panel of bacteria including other porcine mycoplasmas.PCR results were confirmed by dot blot hybridisation.The applicability of the PCR assay to diagnose E.suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs.The E.suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection,thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals.