Tjhie J H, van Kuppeveld F J, Roosendaal R, Melchers W J, Gordijn R, MacLaren D M, Walboomers J M, Meijer C J, van den Brule A J
Department of Clinical Microbiology, Free University Hospital, Amsterdam, The Netherlands.
J Clin Microbiol. 1994 Jan;32(1):11-6. doi: 10.1128/jcm.32.1.11-16.1994.
The sensitivities of three methods of detection of Mycoplasma pneumoniae by a 16S rDNA PCR were compared by using a serial dilution of M. pneumoniae. These methods consisted of a PCR performed directly on cells after a proteinase K pretreatment (direct PCR), a PCR after purification of nucleic acids (DNA-PCR), and a PCR with rRNA sequences as the target after reverse transcription. The direct PCR and the reverse transcription PCR had a sensitivity of 1.5 CFU (approximately 250 genomes). By purification, a 10-fold loss of target DNA occurred, as shown by a 10-fold decrease in sensitivity (15 CFU) of the DNA-PCR. The presence of an excess of human background DNA did not influence the sensitivity of either PCR. The direct PCR was evaluated on samples from patients with respiratory complaints. Direct PCR amplification was possible in 94.9% of the samples, which were tested by amplification of a 326-bp fragment of the beta-globin gene, which was performed to test for the suitability of amplification. Nucleic acid purification was performed on the beta-globin-negative samples, after which only 2% remained negative. A positive correlation between the direct M. pneumoniae PCR and serology, as tested by the microparticle agglutination assay (MAG assay), was found in 88.1% of the cases. A positive MAG assay result was found for samples from 10 (17%) of the patients; samples from 6 (10.2%) of these patients were also positive by PCR. Samples from three patients were found to be positive by the M. pneumoniae PCR and negative by the MAG assay. Persistence of M. pneumoniae, as detected by PCR was observed in three patients. These results indicate that the direct PCR with 16S rDNA could prove to be useful in the detection of M. pneumoniae in respiratory tract samples, although more studies are needed to evaluate the correlation between clinical symptoms and positive test results.
通过对肺炎支原体进行系列稀释，比较了三种基于16S rDNA PCR检测肺炎支原体方法的灵敏度。这些方法包括蛋白酶K预处理后直接对细胞进行PCR（直接PCR）、核酸纯化后进行PCR（DNA-PCR）以及逆转录后以rRNA序列为靶标的PCR。直接PCR和逆转录PCR的灵敏度为1.5 CFU（约250个基因组）。通过纯化，靶标DNA损失了10倍，如DNA-PCR的灵敏度降低10倍（15 CFU）所示。人背景DNA过量的存在并不影响任何一种PCR的灵敏度。对有呼吸道症状患者的样本进行了直接PCR评估。94.9%的样本可进行直接PCR扩增，通过扩增β-珠蛋白基因的326 bp片段来检测样本是否适合扩增。对β-珠蛋白阴性的样本进行核酸纯化，之后只有2%的样本仍为阴性。通过微粒凝集试验（MAG试验）检测发现，直接肺炎支原体PCR与血清学之间在88.1%的病例中呈正相关。10名（17%）患者的样本MAG试验结果为阳性；其中6名（10.2%）患者的样本PCR也呈阳性。发现3名患者的样本肺炎支原体PCR呈阳性而MAG试验呈阴性。通过PCR检测观察到3名患者存在肺炎支原体持续感染。这些结果表明，尽管需要更多研究来评估临床症状与阳性检测结果之间的相关性，但基于16S rDNA的直接PCR可能在呼吸道样本中肺炎支原体的检测中有用。
