Chen X, Miflin J K, Zhang P, Blackall P J
Institute for Animal Husbandry and Veterinary Science, Beijing Academy of Agriculture, People's Republic of China.
Avian Dis. 1996 Apr-Jun;40(2):398-407.
A genomic DNA library of Haemophilus paragallinarum strain Modesto was created. Screening of this library identified four clones that reacted specifically with all 56 isolates of H. paragallinarum tested and failed to react with 24 closely related bacteria from the genera Pasteurella and Actinobacillus. All four clones also failed to react with DNA extracted from one field isolate each of Mycoplasma gallisepticum and Mycoplasma synoviae. The probes based on these four clones were approximately 1.8, 2.3, 3.5, and 5.5 kb in size. The four probes were able to detect between 7.8 and 31.25 ng of purified DNA from homologous strains with no obvious correlation between probe size and sensitivity. The smallest probe, termed P601, was partially sequenced, and primers for two polymerase chain reaction (PCR) tests were designed from these sequence data. Both PCR tests, termed HPG-1 and HPG-2, were shown to be specific, all 41 isolates of H. paragallinarum tested being positive and all 26 non-H. paragallinarum isolates being negative. Both PCR tests were able to detect 1 pg DNA and between 10(2) and 10(3) cells. A method for using the HPG-2 PCR test directly on sinus swabs was developed. Using this method, there was 100% agreement between culture and the direct HPG-2 PCR for the 36 swabs processed. The DNA probes and PCR tests appear to be useful diagnostic tools for the detection of infectious coryza. The tests can be used as confirmatory tests following the isolation of a hemophilic organism. As well, the HPG-2 PCR test appears to be a potential alternative to culture.
构建了副鸡嗜血杆菌莫德斯托菌株的基因组DNA文库。对该文库进行筛选，鉴定出4个克隆，它们与所检测的56株副鸡嗜血杆菌均发生特异性反应，而与巴斯德氏菌属和放线杆菌属的24株密切相关细菌不发生反应。这4个克隆也未与从鸡毒支原体和滑膜支原体的各一株现场分离株中提取的DNA发生反应。基于这4个克隆的探针大小分别约为1.8、2.3、3.5和5.5 kb。这4种探针能够检测到7.8至31.25 ng来自同源菌株的纯化DNA，探针大小与灵敏度之间无明显相关性。最小的探针称为P601，对其进行了部分测序，并根据这些序列数据设计了两种聚合酶链反应（PCR）检测的引物。这两种PCR检测方法，即HPG - 1和HPG - 2，均具有特异性，所检测的41株副鸡嗜血杆菌均为阳性，而26株非副鸡嗜血杆菌分离株均为阴性。两种PCR检测方法均能检测到1 pg DNA以及10²至10³个细胞。开发了一种直接在鼻窦拭子上使用HPG - 2 PCR检测的方法。使用该方法，对36份处理过的拭子进行培养和直接HPG - 2 PCR检测结果完全一致。DNA探针和PCR检测似乎是检测传染性鼻炎的有用诊断工具。这些检测可在分离出嗜血性微生物后用作确证试验。此外，HPG - 2 PCR检测似乎是培养的一种潜在替代方法。
