16S rDNA多重PCR检测牙周组织感染菌及混合感染与慢性牙周炎病变程度关系的研究

占定凤; 刘志伟; 夏肖萍; 胡建成; 陈莉丽; 严杰

Abstract

占定凤,刘志伟,夏肖萍,胡建成,陈莉丽,严杰.16S rDNA多重PCR检测牙周组织感染菌及混合感染与慢性牙周炎病变程度关系的研究[J].Chinese journal of Epidemiology,2005,26(2):120-123

16S rDNA多重PCR检测牙周组织感染菌及混合感染与慢性牙周炎病变程度关系的研究

Study on the detection of P.gingivalis, A.actinomycetemcomitans and T.denticola and the correlation between coinfections of the microbes and levels of chronic periodontitis lesion

Received:March 11, 2004

DOI：

KeyWord: 牙周炎,慢性牙龈卟啉单胞菌放线杆菌齿垢密螺旋体 16S rDNA基因

English Key Word: Periodontitis chronic Porphyromonas gingivalis Actinobacillus Treponema denticola 16S rDNA genes

FundProject:浙江省自然科学基金资助项目(N29953)

Author Name	Affiliation
ZHAN Ding feng	Shenzhen Sanitary and Anti epidemic Station, Shenzhen 518020, China
LIU Zhi wei	Shenzhen Sanitary and Anti epidemic Station, Shenzhen 518020, China
XIA Xiao ping	Shenzhen Sanitary and Anti epidemic Station, Shenzhen 518020, China
HU Jian cheng	Shenzhen Sanitary and Anti epidemic Station, Shenzhen 518020, China
CHEN Li li	浙江大学医学院附属第二医院口腔科
YAN Jie	浙江大学医学院病原生物学教研室

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Abstract:

目的　建立多重 16SrDNAPCR检测慢性牙周炎 (CP)标本中牙龈卟啉单胞菌 (Pg)、伴放线杆菌 (Aa)和齿垢密螺旋体 (Td),了解不同感染情况与CP严重程度的关系。方法　将轻、中和重度15 2例CP患者牙周袋标本和 30名牙周健康者龈沟标本置于 2 0 0 μl裂解缓冲液中,10 0℃水浴 10min后取 10 μl上清液作为聚合酶链反应 (PCR)模板。常规酚氯仿法提取的PgATCC332 77株、AaY4株、TdFM株和E .coliDH5α株DNA分别作为PCR阳性和阴性对照。采用Pg、Aa和Td 16SrDNA特异性引物,建立多重PCR检测上述标本。 3例Pg、Aa和TdPCR结果均阳性标本的目的扩增片段T A克隆后测序。采用 χ² 检验分析不同混合感染情况与牙周炎严重程度之间的关系。结果所建立的多重 16SrDNAPCR最低可检出 10个Pg、2 0个Aa和 2 0个Td细胞。Pg、Aa和Td 16SrDNA扩增片段与已报道的序列比较,相似性分别高达 99.4 5 %、97.0 8%和 96 .5 9%。 30名牙周健康者龈沟标本中,仅有 1名Pg(3.3% )、2名Aa(6 .7% )的 16SrDNA扩增结果阳性,其余标本均阴性。 15 2例CP患者牙周袋标本中,14 7例 (96 .7% )检出Pg、Aa和 /或Td的 16SrDNA,5例 (3.3% )扩增结果均阴性。Pg的PCR检测阳性率 (91.5 %,139 15 2 )明显高于Aa(72 .4 %,110 15 2 )或Td(80 .9%,12 3/152 )(x²=7.07,18.67;P<0.01)0 89.8%的患者标本有上述2种(26.5%)或3种细菌(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(x²=10.43,P<0.01)。结论所建立的多重16S rDNA PCR有较高的敏感性和特异性，可同时检测临床标本Pg,Aa和Tda CP是一种多种病原菌混合感染性疾病，病原菌协同致病作用与CP严重程度有因果关系。

English Abstract:

Objective To establish a 16S rDNA multiplex polymerase chain reaction(PCR) for simultaneously detecting P.gingivalis, A.actinomycetemcomitans and T.denticola in clinical specimens of chronic periodontitis and to study the correlation between different modes of infection and severity of the disease. Methods Periodontal pocket specimens from 152 patients with mild, moderate or advanced chronic periodontitis and gingival sulcus specimens from 30 periodontally healthy individuals were collected and placed in 200 μl lysis buffer. The specimens were incubated in 100℃ for 10 min and 10 μl of the supernatant was directly used as PCR template. DNAs from P.gingivalis strain ATCC33277, A.actinomycetemcomitans strain Y4, T.denticola strain FM and E.coli strain DH5α were used as positive and negative controls in PCR with all of which were prepared by routing phenol chloroform method. A multiplex PCR assay, using three sets of primers specific to 16S rDNA genes of the three anaerobes, was developed to detect the specimens. The target amplification fragments from 3 cases of PCR products positive for all the three anaerobes were sequenced after T A cloning. Chi square test was applied to analyze the correlation between different coinfection of the three anaerobes and severity of the disease. Results The established 16S rDNA multiplex PCR assay was able to detect P.gingivalis, A.actinomycetemcomitans and T.denticola at a minimum of 10, 20 and 20 cells, respectively. In comparison with the reported corresponding sequences, similarities of the nucleotide sequences from the three anaerobes 16S rDNA amplification fragments were as high as 99.45 %, 97.08 % and 96.59 %, respectively. Of the 30 periodontally healthy gingival sulcus specimens, only one( 3.3 %) positive for P.gingivalis and two ( 6.7 %) for A.actinomycetemcomitans could be identified but all of the rest were negative. In the 152 CP periodontal pocket specimens, 147 cases( 96.7 %) were positive for P.gingivalis, A.actinomycetemcomitans and/or T.denticola, respectively, and 5 cases ( 3.3 %) were negative for all the three anaerobes. The positive rate of P.gingivalis detection ( 91.5 %, 139/152 ) was significantly higher than those of A.actinomycetemcomitans ( 72.4 %, 110/152 ) and T.denticola ( 80.9 %, 123/152 )( χ ²= 7.07, 18.67; P 0.01 ). 89.8 % of the specimens from patients showed coinfections with two ( 26.5 %) or three anaerobes ( 63.3 %), and the coinfection rates in the specimens from moderate and advanced CP were remarkably higher than that from mild CP ( χ ²= 10.43, P 0.01 ). Conclusion The 16S rDNA multiplex PCR established in this study showed high sensitivity and specificity, which could be used to detect P.gingivalis, A.actinomycetemcomitans and T.denticola in clinical specimens. CP was an disease caused by multiple pathogenic microbes while the synergistic pathopoiesis of the three microbes was closely related to the severity of the disease.

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