Flemmig T F, Rüdiger S, Hofmann U, Schmidt H, Plaschke B, Strätz A, Klaiber B, Karch H
Department of Operative Dentistry and Periodontics, Julius Maximilian University, Würzburg, Germany.
J Clin Microbiol. 1995 Dec;33(12):3102-5. doi: 10.1128/jcm.33.12.3102-3105.1995.
The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the preferred methods for the detection of A. actinomycetemcomitans in subgingival plaque.
本研究的目的是评估聚合酶链反应（PCR）检测伴放线放线杆菌的敏感性和特异性。将PCR的检测能力与其他三种方法进行了比较：培养增强型PCR（CE-PCR）、菌落杂交（CH）以及采用推测性生化鉴定的传统培养法。使用引物TT-15和TT-16通过PCR扩增伴放线放线杆菌白细胞毒素基因lktA的一段285bp片段。对于CH，将PCR产物用地高辛标记并用作杂交探针。伴放线放线杆菌1D4和1664以及三株临床分离株的PCR产物的核苷酸序列分析显示，受试菌株之间完全同源，与已发表序列相比，仅在第1344位有一个碱基替换。对于人工感染的龈下菌斑，PCR检测伴放线放线杆菌的检测限为每毫升菌斑悬液10³CFU。在PCR（CE-PCR）之前，将龈下菌斑接种在胰蛋白酶大豆血清杆菌肽万古霉素琼脂上培养，可将检测限提高到每毫升10²CFU。对35例牙周病患者和10名牙周健康受试者的龈下菌斑样本分析显示，CE-PCR和CH检测伴放线放线杆菌的总体检出率最高（均为58%），其次是PCR和培养法（均为42%）。以CH作为“金标准”，CE-PCR、PCR和培养法的敏感性分别为88%、65%和58%；特异性分别为84%、89%和79%。当伴放线放线杆菌的患病率在30%至70%之间变化时，CE-PCR提供了可接受的阳性和阴性预测值（≥70%）。单独的PCR在较窄的患病率范围内（30%至50%）提供了相当的预测值，而培养法在任何患病率下都没有提供可接受的预测值。发现PCR和CE-PCR优于采用推测性生化鉴定的培养法，应作为检测龈下菌斑中伴放线放线杆菌的首选方法。
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