藏绵羊子宫内膜炎主要致病菌多重PCR检测方法的建立与评价

Establishment and evaluation of multiplex PCR for detection of main pathogenic bacteria of endometritis in Tibetan sheep

摘要:

本研究旨在建立一种多重PCR方法检测青海藏绵羊子宫内膜炎主要的病原菌。首先，提取5种标准菌株基因组，筛选出特异性引物；然后以标准菌株的基因组为模板，建立多重PCR方法。用无菌棉拭子涂抹藏绵羊子宫，置于LB培养液中培养并编号，48 h后提取样品基因组。运用单一PCR法对600份样品基因组进行检测，记录阳性样品；再挑取单一PCR法检测的阳性样品进行多重PCR检测，再次记录阳性样品，通过计算两种检测方法的符合率验证多重PCR方法；随机挑出30份阳性样品，进行病原菌分离鉴定菌种种类。单一PCR检测的样品中，无乳链球菌感染比例占47.33%，大肠杆菌占34.83%，金黄色葡萄球菌占6.5%，未检出沙门氏菌和化脓隐秘杆菌；多重PCR检测的阳性样品中，无乳链球菌感染比例占45.50%，大肠杆菌占33.50%，金黄色葡萄球菌占6.5%；两种检测结果相比较，多重PCR检测出的符合率均高于95%；分离鉴定的病原菌与两种PCR方法检测出的菌种结果基本一致。成功建立了多重PCR方法并检测出引起青海藏绵羊子宫内膜炎的主要病原菌为无乳链球菌、大肠杆菌和金黄色葡萄球菌。

Abstract:

A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.