慢性牙周炎患者龈下菌斑中伴放线放线杆菌基因型的分析

Genotyping of Actinobacillus actinomycetemcomitans isolated from subgingival plaques of patients with chronic periodontitis

目的 建立龈下菌斑标本中伴放线放线杆菌 (Actinobacillusactinomycetemcomitans,Aa)PCR检测方法 ,了解慢性牙周炎患者不同牙位的龈下菌斑中Aa的感染率及其优势基因型。方法 6 1例慢性牙周炎患者每例采取 2个不同牙位共 12 2份龈下菌斑标本 ,采用培养法分离Aa菌株 ,以PCR或多重PCR检测 16SrDNA基因、lktA基因和fap基因 ,部分扩增产物克隆后测序。结果 在 11例患者的 11份龈下菌斑标本中分离到Aa菌株。 12 2份龈下菌斑中Aa 16SrDNA、lktA和fap检测阳性率分别为 84 .4 %、75 .4 %和 5 0 .0 %。 38.8%的患者 (19 4 9)不同牙位龈下菌斑中检出的Aa基因型不一致。Aa有 4种基因型 ,其优势基因型是 16SrDNA+ lktA+ fap+ ,其次为 16SrDNA+ lktA+ fap- 。部分标本上述 3种基因的扩增片段与文献报道核苷酸序列的同源性为 93.75 %～ 10 0 %。结论 建立的PCR或多重PCR有较高的敏感性和特异性 ,适用于龈下菌斑标本中Aa的快速检测。慢性牙周炎患者Aa感染率较高 ,并存在优势基因型 。

Objective To develop PCR assays for detecting A.actinomycetemcomitans in subgingival plaque samples and to find the infection rate and dominant genotypes of A.actinomycetemcomitans in patients with chronic periodontitis at different tooth sites. Methods 122 subgingival plaque samples were collected from two different tooth sites from each of 61 patients suffering from chronic periodontitis. Culture method was used to isolate A.actinomycetemcomitans strains and PCR or multiplex PCR were applied to detect 16S rDNA, leukotoxin (lktA) and fimbria associated protein (fap) genes of A.actinomycetemcomitans in these samples. Parts of the amplification products were sequenced after cloning. Results 11 strains of A.actinomycetemcomitans were isolated from 11 subgingival plaque samples of 11 patients. In 122 subgingival plaque samples, the positive rates of 16S rDNA, lktA and fap genes by PCR were 84.4%, 75.4% and 50.0% respectively. Different A.actinomycetemcomitans genotypes were detected in different two tooth sites of 19 cases of 49 patients (38.8%). 4 genotypes of A.actinomycetemcomitans were found in these samples and the dominant genotypes were first 16S rDNA +/lktA +/fap + and then 16S rDNA +/lktA +/fap -. The homologies of nucleotide sequence of amplification products from partial samples compared with the reported sequences were from 93.75% to 100%. Conclusion The PCR assays developed in this study with high sensitivity and specificity are suitable for rapid diagnosis of A.actinomycetemcomitans in subgingival plaque samples. A high frequency of A.actinomycetemcomitans infection in the adult periodontitis patients was found, and dominant genotypes of the bacteria in the samples were identified. Some of the patients have been diagnosed as infected with different genotypes of A.actinomycetemcomitans . [