Kamp E M, Bokken G C, Vermeulen T M, de Jong M F, Buys H E, Reek F H, Smits M A
Institute for Animal Science and Health, Lelystad, The Netherlands.
J Vet Diagn Invest. 1996 Jul;8(3):304-9. doi: 10.1177/104063879600800305.
A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific.
建立了一种聚合酶链反应（PCR）检测方法，用于检测从猪采集的鼻拭子和扁桃体拭子样本中的产毒素多杀性巴氏杆菌。用硫氰酸胍和硅藻土分离目标DNA，并同时使用2组源自编码多杀性巴氏杆菌皮肤坏死毒素基因序列的引物。该方法适用于微量滴定板形式，可大规模使用PCR检测。为了识别因扩增失败导致的假阴性检测结果，构建了一个阳性对照模板并添加到每个DNA样本中。用临床样本对PCR检测方法进行评估，并与2种常规用于检测产毒素多杀性巴氏杆菌的方法进行比较：从选择性琼脂上分离以及通过酶联免疫吸附测定（ELISA）直接检测原代培养物提取物中的毒素。用从已知感染产毒素多杀性巴氏杆菌的9个猪群采集的346份鼻拭子和扁桃体拭子样本测试了PCR检测方法的灵敏度。从22份样本中分离出产毒素多杀性巴氏杆菌，ELISA检测中只有28份样本呈阳性，但PCR检测中有40份呈阳性；因此，PCR检测是这3种方法中最灵敏的。用从6个经认证无产毒素多杀性巴氏杆菌的猪群采集的372份拭子样本测试了PCR检测方法的特异性。这些样本中均未分离出产毒素多杀性巴氏杆菌，ELISA检测全部呈阴性，PCR检测中有370份呈阴性。2份阳性样本来自同一窝的2头猪，在PCR检测中仅呈弱阳性。从这些结果得出结论，PCR检测方法不仅高度灵敏而且高度特异。
