Baumeister A K, Runge M, Ganter M, Feenstra A A, Delbeck F, Kirchhoff H
Institut für Mikrobiologie und Tierseuchen, Tierärztliche Hochschule Hannover, Germany.
J Clin Microbiol. 1998 Jul;36(7):1984-8. doi: 10.1128/JCM.36.7.1984-1988.1998.
In the present investigation we developed a method for the detection of Mycoplasma hyopneumoniae in bronchoalveolar lavage fluid (BALF) of pigs by PCR with a primer pair flanking a DNA fragment of 853 bp specific for M. hyopneumoniae. Several methods were tested to eliminate the amplification inhibitors present in BALFs. The best results were obtained by the extraction of the DNA from the BALFs. By the PCR performed with the extracted DNA, 10(2) CFU of M. hyopneumoniae could be detected in 1 ml of BALF from specific-pathogen-free swine experimentally inoculated with M. hyopneumoniae. DNA from 11 other mycoplasma species and 17 cell-walled bacterial species colonizing the respiratory tracts of pigs was not amplified. In a field study BALFs from 40 pigs from farms with a history of chronic pneumonia were tested for M. hyopneumoniae by cultivation and by PCR (i) with BALFs incubated in Friis medium and (ii) with DNA extracted from the BALFs. In addition, PCR was performed with postmortem lung washings from 19 of the 40 pigs, and immunofluorescence tests were carried out with sections of lungs from 18 of the 40 pigs. M. hyopneumoniae could not be detected in 18 of the 40 pigs by any of the five methods tested. The remaining 22 pigs showed a positive reaction by the PCR with DNA extracted from the BALFs and variable positive reactions by the other tests. A complete correspondence could be observed between the immunofluorescence test result and the result of PCR with DNA. The investigation shows that the PCR with DNA extracted from BALFs is a suitable technique for the sensitive and specific in vivo detection of M. hyopneumoniae.
在本研究中，我们开发了一种通过PCR检测猪支气管肺泡灌洗液（BALF）中猪肺炎支原体的方法，所用引物对侧翼为猪肺炎支原体特异的853 bp DNA片段。测试了几种方法以消除BALF中存在的扩增抑制剂。从BALF中提取DNA获得了最佳结果。用提取的DNA进行PCR，在1 ml经实验接种猪肺炎支原体的无特定病原体猪的BALF中可检测到10(2) CFU的猪肺炎支原体。来自其他11种支原体和17种定植于猪呼吸道的细胞壁细菌的DNA未被扩增。在一项现场研究中，对来自有慢性肺炎病史猪场的40头猪的BALF进行了猪肺炎支原体培养检测以及PCR检测，其中PCR检测分别采用（i）在弗里斯培养基中孵育的BALF和（ii）从BALF中提取的DNA。此外，对40头猪中的19头进行了死后肺冲洗液的PCR检测，并对40头猪中的18头进行了肺组织切片免疫荧光检测。在所测试的五种方法中，40头猪中有18头未检测到猪肺炎支原体。其余22头猪通过从BALF中提取DNA进行PCR检测呈阳性反应，而其他检测呈不同程度的阳性反应。免疫荧光检测结果与DNA PCR检测结果完全一致。该研究表明，用从BALF中提取的DNA进行PCR是一种灵敏且特异的体内检测猪肺炎支原体的合适技术。
