Goncharoff P, Figurski D H, Stevens R H, Fine D H
School of Dental and Oral Surgery, Division of Oral Infectious Diseases, Columbia University, New York 10032.
Oral Microbiol Immunol. 1993 Apr;8(2):105-10. doi: 10.1111/j.1399-302x.1993.tb00554.x.
Actinobacillus actinomycetemcomitans has been strongly implicated in the etiology of localized juvenile periodontitis. Techniques used in the identification of this periodontal pathogen include cultural, biochemical, immunological and DNA hybridization analysis. In this study, we report the use of polymerase chain reaction (PCR) to amplify unique sequences of A. actinomycetemcomitans. Specific oligonucleotide primers LKT2 and LKT3 were designed to hybridize to the A. actinomycetemcomitans lktA gene, which encodes leukotoxin, a putative A. actinomycetemcomitans virulence factor. The LKT2 and LKT3 primers amplified lktA-specific sequences from all 12 A. actinomycetemcomitans strains tested. In another set of experiments, 13 other bacterial species, most of which are normal residents of the oral cavity, were tested with these primers. These PCR amplifications also contained 2 additional primers, RRN4 and RRN5, which served as positive controls; RRN4 and RRN5 were designed to amplify specific sequences of eubacterial 16S ribosomal DNA (rDNA). PCR amplifications of all bacterial species tested, including A. actinomycetemcomitans, yielded 16S rDNA-specific DNA fragments. Furthermore, each bacterial species tested, with the exception of A. actinomycetemcomitans, failed to amplify lktA sequences. The LKT and RRN primers were used in further PCR experiments to detect A. actinomycetemcomitans directly from gingival fluid samples. The results clearly demonstrate the simplicity, rapidity, specificity and accuracy of the LKT primers in the identification of A. actinomycetemcomitans.
伴放线放线杆菌与局限性青少年牙周炎的病因密切相关。用于鉴定这种牙周病原体的技术包括培养、生化、免疫和DNA杂交分析。在本研究中，我们报告了使用聚合酶链反应（PCR）来扩增伴放线放线杆菌的独特序列。设计了特异性寡核苷酸引物LKT2和LKT3，使其与伴放线放线杆菌的lktA基因杂交，该基因编码白细胞毒素，这是一种假定的伴放线放线杆菌毒力因子。LKT2和LKT3引物从所有12株受试的伴放线放线杆菌菌株中扩增出lktA特异性序列。在另一组实验中，用这些引物对另外13种细菌进行了检测，其中大多数是口腔的正常定植菌。这些PCR扩增还包含另外2种引物RRN4和RRN5，它们作为阳性对照；RRN4和RRN5被设计用于扩增真细菌16S核糖体DNA（rDNA）的特异性序列。对包括伴放线放线杆菌在内的所有受试细菌进行的PCR扩增均产生了16S rDNA特异性DNA片段。此外，除伴放线放线杆菌外，每种受试细菌均未能扩增出lktA序列。LKT和RRN引物被用于进一步的PCR实验，以直接从龈沟液样本中检测伴放线放线杆菌。结果清楚地证明了LKT引物在鉴定伴放线放线杆菌方面的简便性、快速性、特异性和准确性。
