To establish the multiplex PCR with 16S ribosomal DNA amplification for the simultaneous detection of P.gingivalis,A.actinomycetemcomitans and T.denticola in clinical specimens of patients with chronic periodontitis and to determine the correlation between mixed infection and the severity of disease,152 cases of patients with chronic periodontitis,classified by method of Hugoson as mild,moderate and severe cases,and 30 periodontally healthy individuals as normal control were chosen for study.The periodontal pocket specimens of patients and the gingival sulcus specimens of normal controls were collected,placed in 200 μl of lysis buffer,incubated at 100℃ for 10 minutes and 10μl of the supernatant were taken as PCR template.DNAs from P.gingivalis(strain ATCC33277),A.actinomycetemcomitans(strain Y4) and T.denticola(strain FM) were used as the positive controls;and the DNA from E.coli(strain DH15) was used as the negative control.for PCR.These DNAs were extracted by phenol-chloroform extraction method A multiplex PCR assay by using these 3 sets of primers specific to 16S ribosomal DNA genes of these 3 anaerobes was established for the simultaneous detection of these 3 microorganisms in the clinic specimens The target amplification fragments from 3 cases with PCR products positive for all the 3 anaerobes were sequenced after T-A cloning.It was found that the minimal amount of DNA detected by this established multiplex PCR assay was equivalent to that of 10 cells of P.gingivalis,20 cells of A actinomycetemcimitans or 20 cells of T.denticola.In comparison with the reported corresponding sequences,the similarities of nucleotide sequences of the 16S ribosomal DNA amplified fragments of these 3 anaerobes were as high as 99.45%,97.08% and 96.59% respectively.In the gingival sulcus specimens of 30 periodonally heaalthy individuals,there were only one (3.3%) positive case for P.gingivalis and 2 (6.7%) positive cases for A actinomycetemcomitans,and the rests were all negative.However,of the periodonal pocket specimens of 152 patients with chronic periodontitis,147 cases (96.7%) were positive and 5 cases (3.3%) were negative for all these 3 anaerobes.The positive rate of P.gingivalis(91.5%) was significantly higher than those of A.actinomycetemcomitans (72.4%) and T.denticola(80.9%).In 89.8% of the specimens from patients,co-infections with two (26.5%) or three (63.3%) anaerobes were evident and the co-infection rates in the specimens from moderate and severe cases of chronic periodontiis were remarkably higher than that from mild cases.It concludes that the multiplex PCR with 16S ribosomal DNA amplification established in this study shows high sensitivity and specificity,that it can be used in clinics as a laboratory method to detect thee presence of P.gingivalis,A.actinomycetemcomitans and T.denticola,and it proves that chronic periodontitis is a disease caused by multiple pathogenic microbes and the synergistic pathogenicity of these three anaerobes is closely related with the severity of disease.%目的 建立多重16S rDNA PCR方法用以同时检测慢性牙周炎(CP)临床标本中牙龈卟啉单胞菌(Pg)、伴放线放线杆菌(Aa)和齿垢密螺旋体(Td)，并了解不同感染情况与慢性牙周炎严重程度的关系。方法 采集152例CP患者牙周袋标本，并按Hugoson的方法将其分为轻、中和重度三类，另采集30例牙周健康者龈沟标本作为正常对照。上述临床标本置于200μl裂解缓冲液中，100℃冰浴10 min后取10μl上清液直接作为PCR的模板。采用常规酚-氯仿法提取Pg ATCC33277株、Aa Y4株、Td FM株和E.coli DH5α株的DNA分别作为PCR的阳性和阴性对照。采用Pg、Aa和Td 16S rDNA特异性引物，建立多重PCR检测上述标本。3例Pg、Aa和Td PCR结果均阳性患者牙周袋标本目的扩增片段T-A克隆后测序。采用χ2检验分析Pg、Aa和/或Td感染率与牙周炎严重程度之间的关系。结果 所建立的多重16S rDNA PCR最低可检出10个Pg、20个Aa和20个Td细胞。Pg、Aa和Td 16S rDNA扩增片段测序结果与已报道的相应序列比较，相似性分别高达99.45％、97.08％和96.59％。30例牙周健康者龈沟标本中，仅有1例(3.3％)Pg、2例(6.7％)Aa的16S rDNA扩增结果阳性，其余标本均阴性。152例CP患者牙周袋标本中，147例(96.7％)检出Pg、Aa和/或Td的16S rDNA，5例(3.3％)扩增结果均阴性。Pg的PCR检测阳性率(91.5％，139/152)明显高于Aa(72.4％，110/152)或Td(80.9％，123/152)(χ2=7.07，18.67；P＜0.01)。89.8％的患者标本有两种(26.5％)或三种微生物(63.3％)同时感染，且中、重度牙周炎混合感染率明显高于轻度牙周炎(χ2=10.43，P＜0.01)。结论 所建立的同时检测多重16S rDNA PCR有较高的敏感性和特异性，可用于临床Pg、Aa和Td实验室检测。多种牙周病原微生物协同致病作用与CP严重程度有因果关系。
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