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首页> 外文期刊>Journal of Clinical Microbiology >Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection ofActinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis
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Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection ofActinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis

机译：使用保守的和物种特异性的16S rRNA基因引物改进的多重PCR，用于同时检测放线放线杆菌，连翘拟杆菌和牙龈卟啉单胞菌

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Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, andPorphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and fourP. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species.A. actinomycetemcomitans, B. forsythus, andP. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays.
机译：在推定的牙周病原体中，最有说服力的是，放线放线杆菌（Actinobacillus actinomycetemcomitans），连翘拟杆菌（Bacteroides forsythus）和牙龈卟啉单胞菌（emorphyromonas gingivalis）是牙周炎的病因。因此，用于检测这三个物种的技术将很有价值。我们之前发布了检测 A的多重PCR的说明。放线菌和 P。牙龈炎。本论文提出了对该技术的改进，该技术现在允许对所有三种牙周病原体进行更灵敏的检测。敏感性是通过测试连续稀释的emA来确定的。放线菌， B。连翘和 P。牙龈细胞针对（i）GenBank-EMBL数据库中的所有基因序列，（ii）六个 A测试了引物特异性。放线菌 ，一个 B。连翘和四个 P。牙龈炎菌株，（iii）八种不同的口腔细菌，以及（iv）来自20位健康受试者的龈上和龈下菌斑样品，以及来自10位牙周炎患者的龈下菌斑样品。多重PCR的检测极限为10A。放线菌， P 10。牙龈炎和100 B。连翘细胞。通过以下事实证实了特异性：（i）我们的正向引物均不与其他口腔物种的16S rRNA基因同源;（ii）对所有 A检测到预测大小的扩增子。放线菌， B。连翘和 P。测试了牙龈菌菌株，以及（iii）没有检测到其他八种细菌的扩增子。放线菌， B。连翘和 P。从牙周健康的龈上菌斑样品中分别检出20个中的6个，20个中的1个和20个中的11个，在牙周健康样本中分别检出20个中的4个，20个中的7个，20个中的13个以及20个中的13个科目。在牙周炎患者中，分别在10个样本中的7个，10个样本中的10个和10个样本中的7个中检测到了这种生物。与常规PCR分析相比，同时检测三种牙周病原体是该技术的优势。

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