Hewinson R G, Griffiths P C, Bevan B J, Kirwan S E, Field M E, Woodward M J, Dawson M
Department of Bacteriology, Central Veterinary Laboratory, Addlestone, Surrey, UK.
Vet Microbiol. 1997 Feb;54(2):155-66. doi: 10.1016/s0378-1135(96)01268-0.
A polymerase chain reaction (PCR) assay was developed to detect. Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5' non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.
开发了一种聚合酶链反应(PCR)检测方法,用于检测禽类粪便和组织样本中的鹦鹉热衣原体DNA。设计引物以扩增一个264 bp的产物,该产物来源于编码主要外膜蛋白的ompA基因5'非翻译区的一部分和编码区的一部分。使用内部探针通过Southern杂交确认扩增序列。发现联合检测的灵敏度在60至600 fg衣原体DNA(约6至60个基因组拷贝)之间。该检测方法的特异性得到了证实,因为从含有几种沙眼衣原体血清型、肺炎衣原体菌株、猪衣原体标准菌株的样本中,以及从含有禽类肠道菌群中常见微生物的样本中均未获得PCR产物。在本研究中,对中央兽医实验室在6个月期间收到的404份禽类粪便和141份禽类组织样本进行了PCR、抗原检测ELISA分析,并在可能的情况下进行了细胞培养分离。与ELISA和细胞培养相比,或仅与ELISA相比,PCR表现良好。PCR检测方法特别适合检测禽类粪便样本中的鹦鹉热衣原体DNA。当应用于与人类鹦鹉热病例相关的小型接触禽类的组织样本时,该检测方法也很有用,因为ELISA结果为阴性,且由于需要快速诊断,衣原体分离是一种不太理想的方法。