Polymerase Chain Reaction Analysis of the Clonality of Porphyromonas Gingivalis and Collagenase Gene

目的 :本研究旨在分析牙龈卟啉菌基因型和其重要毒力因子胶原酶基因 (collagenasegene ,PrtC)的遗传异质性 ,了解细菌遗传变异与其对牙周致病性的相关性。方法 :采用随机引物聚合酶链反应 (AP -PCR)的方法 ,对从 2 4例牙周炎患者口腔中分离的 79株牙龈卟啉菌和参考菌株ATCC332 77进行DNA指纹分析。并随机选择 2 4株临床菌株 ,进一步检测是否存在特异性的胶原酶基因片段。对照菌株为伴放线放线杆菌Y4和中间普氏菌ATCC2 5 6 11,通过酶切鉴定和DNA序列分析 ,了解PrtC基因遗传多样性的变化。结果 :80株牙龈卟啉菌共获得 7种基因型 (I—VII) ,以VII所占比例最高 (2 5 .8%)。 2 4株临床菌株和ATCC332 77中均扩增出特异性的胶原酶基因片段 (5 48bp)。将 4株细菌的序列分析结果与国外的报道相比较 ,发现一些基因序列存在差异 ,其中有 6个核苷酸碱基缺失。结论 :临床分离的牙龈卟啉菌中可检测到多种基因型 ,存在明显的个体差异 ,这些菌株具有合成胶原酶的能力 ,不同菌株胶原酶基因之间存在遗传异质性的变化。

Objective:Porphyromonas gingivalis is associated with the initiation and progression of adult periodontitis.The clonality and virulence of P.gingivalis have been shown to exhibit genetic diversity possibily resulting in variation of virulence.The aim of the present study was to investigate the genotypic characterization of P.gingivalis and the heterogeneity of a potential virulence factor-collagenase.Methods:Arbitrarily primed polymerase chain reaction (AP-PCR) was applied to 80 P.gingivalis strains, including reference strain P.gingivalisATCC33277 and 79 clinical strains isolated from 24 unrelated Chinese periodontitis patients.The middle portion of PrtC gene from 24 P.gingivalis isolates was amplified by PCR and the PCR products were digested with restriction endonucleases PstI and PvuI.To evaluate the sequence heterogeneity of the P.gingivalis prtC genes,sequence analysis of four prtC gene of clinical isolates was performed.Results:Random primer OPA-05 and OPA-17 distinguished 7 AP-PCR profiles(ⅠthroughⅦ). The majority of the strains belonged to type Ⅶ which accounted for 25.8%. A 548bp fragment of prtC gene was detected from 24 clinical strains.The restriction analysis of the PCR products revealed fragment patterns identical to the known sequence.All analyzed genes were heterogeneous in their nucleotide composition and differed from reference strain P.gingivalis 53977.Six nucleotides deletion in the sequence of clinical strains were found.Conclusion:The results demonstrated genetic diversity existed among these clinical strains isolated from Chinese periodontitis patients and the prtC genes are heterogeneous in their nucleotide sequence.