Schoeb T R, Dybvig K, Keisling K F, Davidson M K, Davis J K
Department of Pathobiology, University of Florida, Gainesville 32610, USA.
J Clin Microbiol. 1997 Jul;35(7):1667-70. doi: 10.1128/jcm.35.7.1667-1670.1997.
To improve the detection of Mycoplasma pulmonis contamination of isolates of cilia-associated respiratory (CAR) bacillus, we developed a nested PCR method using primers for 16S rRNA gene sequences. Of 140 samples of 16 different CAR bacillus isolates, 73 (52%) were inhibitory in the first PCR, as indicated by the absence of amplicons of the internal control, but only 11 of 140 (7.9%) were inhibitory in the second PCR. Of 27 samples known to contain M. pulmonis, only 12 (44%) were positive in the first PCR, but 25 of 27 (93%) were positive in the second PCR. Nested PCR also detected M. pulmonis in 21 of 61 (34%) CAR bacillus samples from which M. pulmonis could not be cultured and identified 2 additional M. pulmonis-contaminated CAR bacillus isolates. Of 359 respiratory and reproductive tract lavage samples from rats and mice, 35 (9.8%) were inhibitory in the first PCR, but only 15 (4.2%) were inhibitory in the second PCR. Of 72 lavage specimens from rats inoculated with an avirulent, poorly infective M. pulmonis strain, 14 (19%) were positive by nested PCR, but only 2 of 72 (2.8%) were positive by culture. Nested PCR also detected M. pulmonis in 14 of 20 (70%) paraffin sections of lung and trachea from rats and mice inoculated with CAR bacillus isolates known to contain M. pulmonis, whereas single PCR gave no positive results. We conclude that nested PCR is superior to single PCR or culture for detecting M. pulmonis, and that M. pulmonis is present in all but four CAR bacillus isolates in our collection that were from naturally infected rats; the four isolates that were exceptions were obtained from rats from a single colony.
为提高对纤毛相关呼吸道（CAR）杆菌分离株中肺炎支原体污染的检测能力，我们开发了一种巢式PCR方法，该方法使用针对16S rRNA基因序列的引物。在16种不同CAR杆菌分离株的140个样本中，73个（52%）在第一次PCR中具有抑制性，表现为内部对照无扩增子，但在第二次PCR中，140个样本中只有11个（7.9%）具有抑制性。在已知含有肺炎支原体的27个样本中，第一次PCR只有12个（44%）呈阳性，但第二次PCR中27个样本中有25个（93%）呈阳性。巢式PCR还在61个CAR杆菌样本中的21个（34%）中检测到肺炎支原体，这些样本无法培养出肺炎支原体，并鉴定出另外2株被肺炎支原体污染的CAR杆菌分离株。在来自大鼠和小鼠的359份呼吸道和生殖道灌洗样本中，35个（9.8%）在第一次PCR中具有抑制性，但在第二次PCR中只有15个（4.2%）具有抑制性。在接种了无毒、感染性差的肺炎支原体菌株的大鼠的72份灌洗标本中，巢式PCR检测出14个（19%）呈阳性，但培养法检测出72个样本中只有2个（2.8%）呈阳性。巢式PCR还在接种了已知含有肺炎支原体的CAR杆菌分离株的大鼠和小鼠的20个肺和气管石蜡切片中的14个（70%）中检测到肺炎支原体，而单次PCR未得到阳性结果。我们得出结论，巢式PCR在检测肺炎支原体方面优于单次PCR或培养法，并且在我们收集的来自自然感染大鼠的CAR杆菌分离株中，除了4株外，其余均存在肺炎支原体；这4株例外分离株来自单个菌落的大鼠。
