实时荧光定量PCR法检测治疗后龈下菌斑中优势菌的变化

上海口腔医学 ›› 2013, Vol. 22 ›› Issue (3): 297-301.

实时荧光定量PCR法检测治疗后龈下菌斑中优势菌的变化

邓淑丽¹，王颖¹，何佳燕²，陈卓¹，陈晖¹

(1.浙江大学医学院附属口腔医院牙体牙髓病科，浙江杭州 310006；2.浙江大学农业与生物技术学院，浙江杭州 310003)

收稿日期:2012-12-13 修回日期:2013-01-08 出版日期:2013-06-10 发布日期:2013-06-10
通讯作者: 陈晖,Tel:0571-87217437,E-mail:Huic66@hotmail.com
作者简介:邓淑丽(1971- )，女，硕士，副主任医师，E-mail:dengshuli3@163.com
基金资助:
2011年国家临床重点专科建设项目；浙江省自然科学基金(LY13H140002)；浙江省教育厅基金(20061258)；浙江省医药卫生一般研究计划(2012KYB121)

Quantitative real-time PCR for target periodontal bacteria in subgingival plaque before and after local delivery of periocline, scaling and root planning

DENG Shu-li¹, WANG Ying¹, HE Jia-yan², CHEN Zhuo¹, CHEN Hui¹

1. Department of Conservative Dentistry, Affiliated Hospital of Stomatology，College of Medicine, Zhejiang University. Hangzhou 310006; 2. Institute of Agriculture and Biological Technology, Zhejiang University. Hangzhou 310003, Zhejiang Province, China

Received:2012-12-13 Revised:2013-01-08 Online:2013-06-10 Published:2013-06-10
Supported by:
Supported by 2011 National Clinical Specialist Construction Project; Natural Science Foundation of Zhejiang Province(LY13H140002); Education Department Funds of Zhejiang Province(20061258) and Medical General Research Project of Zhejiang Province(2012KYB121).

摘要/Abstract

摘要： 目的：运用实时荧光定量PCR(qRT-PCR)技术检测慢性牙周炎患者治疗前、后龈下菌斑中牙龈卟啉单胞菌(Pg)和中间普氏菌(Pi)的拷贝数，以了解qRT-PCR对2种牙周致病菌检测的敏感性和牙周治疗的疗效。方法：选取62例中、重度牙周炎患者，分别采用龈下刮治、牙周袋内盐酸米诺环素软膏(派丽奥)给药和两者综合治疗，采集治疗前、后(7d)龈下菌斑，提取细菌基因组DNA，合成针对Pg和Pi的16S rRNA基因的特异引物，运用qRT-PCR法检测Pg和Pi的拷贝数。采用SAS 9.1.3软件包对数据进行Kruskal-Wallis和 Wilcoxon 秩和检验。结果：Pg、Pi在不同样品组中检测到的绝对拷贝数数量分别为103～106和102～106，在慢性牙周炎患者龈下菌斑中的检出率和绝对拷贝数量显著高于健康对照组(P<0.05)；3组治疗后的Pg数量比治疗前下降，其中综合治疗组和派丽奥治疗组下降率显著高于龈下刮治组。Pi的数量在派丽奥治疗组和龈下刮治组治疗后无显著减少，但在综合治疗组显著下降(P<0.05)。结论：qRT-PCR法能快速鉴定和精确定量Pg和Pi；综合治疗法比单一疗法能更有效抑制Pg和Pi。

关键词: 实时荧光定量PCR, 牙龈卟啉单胞菌, 中间普氏菌

Abstract: PURPOSE: To compare the copy number of Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi) in subgingival plaque before and after local delivery of periocline (2% minocycline hydrochloride ointment, MO), scaling and root planning (SRP) by quantitative real-time PCR (qRT-PCR) and evaluate the efficacy. METHODS: Sixty-two adults with moderate to severe chronic periodontitis were selected in the study. Microbial samples were taken from pocket before and after MO and SRP(7d). The samples were evaluated by qRT-PCR for Pg and Pi. Microbiological effectiveness of treatments was assessed using Kruskal-Wallis and Wilcoxon rank-sum test. All tests were two-sided with a significance level of 0.05. All analyses were conducted with SAS 9.1.3 software package. RESULTS: The copy number of Pg and Pi in subgingival plaque was 103-106 and 102-106. Bacterial loads of Pg were reduced in SPR+ MO, SRP and MO site. The counts of Pi decreased in SRP+ MO sites compared with those in the MO or SRP alone sites significantly (P<0.05). CONCLUSIONS: Quantitative real-time PCR (qRT-PCR) is used as a powerful tool with high sensitivity and specificity to quantitatively assess target periodontal bacteria. The results show that subgingival administration of MO and SRP was effective for reducing pathogenic bacteria and improving clinical outcome.

Key words: Quantitative real-time PCR, Porphyromonas gingivalis, Prevotella intermedia

中图分类号:

R781.42

邓淑丽, 王颖, 何佳燕, 陈卓, 陈晖. 实时荧光定量PCR法检测治疗后龈下菌斑中优势菌的变化[J]. 上海口腔医学, 2013, 22(3): 297-301.

DENG Shu-li, WANG Ying, HE Jia-yan, CHEN Zhuo, CHEN Hui. Quantitative real-time PCR for target periodontal bacteria in subgingival plaque before and after local delivery of periocline, scaling and root planning[J]. Shanghai Journal of Stomatology, 2013, 22(3): 297-301.

参考文献

[1] Haffajee AD, Socransky SS. Microbial etiological agents of destructive periodontal diseases [J]. Periodontol 2000,1994,5(1):78-111.
[2] Sakellari D, Goodson JM, Kolokotronis A, et al. Concentration of 3 tetracyclines in plasma, gingival crevice fluid and saliva[J]. J Clin Periodontol, 2000, 27(1): 53-60.
[3] 李晓燕, 王宏, 石云凯, 等. 米诺环素局部应用对洁治和根面平整影响的Meta分析[J]. 口腔医学研究, 2011, 27(9): 817-820.
[4] 马晓丽, 康宏, 龙飞, 等. 局部应用2%米诺环素软膏治疗慢性牙周炎疗效的系统评价[J].实用口腔医学杂志, 2010, 26(4): 507-511.
[5] Boutaga K, Van Winkelhoff AJ, Vandenbroucke-grauls CM, et al. The additional value of real-time PCR in the quantitative detection of periodontal pathogens[J]. J Clin Periodontol, 2006, 33(6): 427-433.
[6] Tomazinho LF, Avila-Campos MJ. Detection of Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens in chronic endodontic infection[J]. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2007, 103(2): 285-288.
[7] Nadkarni MA, Martin FE, Jacques NA, et al. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set[J]. Microbiology, 2002, 148(Pt 1): 257-266.
[8] Morikawa M, Chiba T, Tomii N, et al. Comparative analysis of putative periodontopathic bacteria by multiplex polymerase chain reaction [J]. J Periodont Res, 2008, 43(3): 268-274.
[9] Kuboniwa M, Amano A, Kimura KR, et al. Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes[J]. Oral Microbiol Immunol, 2004, 19(3): 168-176.
[10] Merchant AT, Pitiphat W. Researching periodontitis: challenges and opportunities[J]. J Clin Periodontol, 2007, 34(12): 1007-1015.
[11] Deng S, Jepsen S, Dommisch H, et al. Cysteine proteases from Porphyromonas gingivalis and TLR ligands synergistically induce the synthesis of the cytokine IL-8 in human artery endothelial cells [J]. Arch Oral Biol, 2011, 56(12): 1583-1591.
[12] Mccoll E, Patel K, Dahlen G, et al. Supportive periodontal therapy using mechanical instrumentation or 2% minocycline gel: a 12 month randomized, controlled, single masked pilot study [J]. J Clin Periodontol, 2006, 33(2): 141-150.
[13] Eguchi T, Koshy G, Umeda M, et al. Microbial changes in patients with acute periodontal abscess after treatment detected by Pado Test[J]. Oral Dis, 2008, 14(2): 180-184.
[14] Shoemaker NB, Vlamakis H, Hayes K, et al. Evidence that extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon[J]. Appl Environ Microbiol, 2001, 67(2): 561-568.
[15] Takahashi N, Ishihara K, Kimizuka R, et al. The effects of tetracycline, minocycline, doxycycline and ofloxacin on Prevotella intermedia biofilm[J]. Oral Microbiol Immunol, 2006, 21(6): 366-371.
[16] Fosse T, Madinier I, Hitzig C, et al. Prevalence of β-lactamase-producing strains among 149 anaerobic gram-negative rods isolated from periodontal pockets[J]. Oral Microbiol Immunol, 1999, 14(6): 352-357.
[17] Maeda H, Fujimoto C, Haruki Y, et al. Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria[J]. FEMS Immunol Med Microbiol, 2003, 39(1): 81-86.