实验大鼠泰泽菌检测方法的比较研究

目的　比较实验大鼠泰泽菌检测方法─nested PCR、IFA、免疫抑制诱发试验 触片染色镜检和组织病理学诊断。方法　根据泰泽菌 16SrDNA合成引物 ,对 16个菌株作nested PCR扩增并进行特异性、敏感性试验、验证。将此PCR应用于 2 0只免疫抑制Wistar大鼠和 5只非免疫抑制SD大鼠泰泽菌检测 ,并作IFA、常规细菌学检测和组织病理学诊断。结果　nested PCR中仅有泰泽菌出现 196bp特异性扩增条带 ;而 15株非泰泽菌均未出现此扩增条带。该PCR能检出 10pg泰泽菌DNA。将此PCR应用于大鼠泰泽菌检测 ,结果未检出阳性样品。nested PCR与常规细菌学检测、组织病理学诊断结果相一致。采用IFA方法 ,以购得的大鼠泰泽菌抗原片对上述 2 5份大鼠血清进行检测 ,结果有 6份血清产生非特异性反应。结论　采用IFA对动物群进行筛查出现阳性结果 ,须采用免疫抑制诱发试验、PCR和组织病理学诊断技术组合进一步验证。本研究建立的nested PCR方法 ,特异、敏感、快速 ,结合组织病理学诊断技术对实验动物泰泽菌感染可做出精确诊断。

Comparative Study on Detecting Clostridum Piliforme Infections in Laboratory Rats

Objective To evaluate the usefulness of nested-polymerase chain reaction (nested-PCR), indirect fluorescent antibody assay (IFA), immunosuppressive provocation tests, and histopathological diagnosis assays for detecting Clostridum Piliforme (C. Piliforme) infections in laboratory rats. Methods In this study, the nested-PCR primers, which we synthesized to amplify C. Piliforme 16S ribosomal DNA (rDNA) sequences as an identification system for this organism, were performed by using C. Piliforme and 15 non-C. Piliforme strains. Specimens taken from 20 immunosuppressive Wistar rats and 5 non-immunosuppressive SD rats and submitted to detect C. Piliforme by nested-PCR. Results The C. Piliforme reference strain revealed positive result indicated by a 196 bp specific fragment on gel, while 15 non-C. Piliforme standard strains gave negative results demonstrating that the inner primers should be specific for C. Piliforme tested. The nested-PCR would be able to detect as little as 10 pg C. Piliforme-DNA. Tissues samples taken from rats and submitted to detect C. Piliforme by nested-PCR were found negative results, and the results of nested-PCR and consistent with those of conventional bacteria identification tests and histopathological diagnosis assays. 25 serum samples taken from rats were submitted to detect C. Piliforme antibodies by IFA were found heterologous cross-reactive antigens such as some strains of Clostridia and Bacillus. Conclusion IFA is best used for colony surveillance, and follow-up testing may include immunosuppressive provaction tests. Confirmation of C. Piliforme infections is best performed by PCR, bacteria identification tests, and histopathological diagnosis assays. In our study, the results of nested-PCR are consistent with those of bacteria identification tests and histopathological diagnosis assays. The data have also demonstrated that the nested-PCR established here is a quick, reliable, sensitive and specific method for detecting C. Piliforme infections in laboratory rats.