巢式-实时PCR检测梅毒初诊患者不同样本中梅毒螺旋体靶DNA

中华皮肤科杂志 ›› 2017, Vol. 50 ›› Issue (5): 346-350.

巢式-实时PCR检测梅毒初诊患者不同样本中梅毒螺旋体靶DNA

叶兴东¹,高方铭²,曹文苓³,林宏达⁴,任泽舫⁴

1. 广州市皮肤病防治所
2. 广州市皮肤病防治所&广州医科大学皮肤病研究所
3. 广州市皮肤病防治研究所
4. 中山大学公共卫生学院

收稿日期:2017-02-10 修回日期:2017-02-27 发布日期:2017-04-28
通讯作者: 叶兴东 E-mail:xingdongye@21cn.com
基金资助:
广州地区梅毒苍白螺旋体 arp/tpr 基因分子流行病学及梅毒控制策略研究;广州市梅毒流行病学及预防控制研究;广州地区梅毒螺旋体感染不同人群危险因素分析及其耐药性研究;广州地区梅毒螺旋体感染不同人群危险因素分析及其耐药性研究;广州地区梅毒苍白螺旋体 arp/tpr 基因分子流行病学及梅毒控制策略研究

Application of nested real?time PCR in detecting Treponema palladium DNA in various clinical samples from patients preliminarily diagnosed as syphilis

Received:2017-02-10 Revised:2017-02-27 Published:2017-04-28
Contact: xingdong ye E-mail:xingdongye@21cn.com
Supported by:
广东省科技计划项目;广州市医药卫生重大项目;广州市科技攻关项目;广州市科技攻关项目;广东省科技计划项目

摘要/Abstract

摘要： 目的探讨巢式-实时PCR（NR?PCR）检测初诊梅毒患者不同样本中梅毒螺旋体（Tp）靶DNA的可行性及应用前景。方法以Tp polA为靶基因，用NR?PCR检测梅毒患者初诊时皮损拭子、血清、全血、脑脊液、末梢耳垂血等样本中的靶DNA，用统计软件SPSS.13分析结果。结果 NR?PCR检测Tp polA靶DNA的极限为2个Tp/ml，总体阳性率为71.7%，检测不同类样本的阳性率从高到低依次为：耳垂血92.0% > 脑脊液90.2% > 拭子74.3% > 血清66.9% > 全血64.2%。NR-PCR结果与血清学检查结果的一致性为76.0%（152/200）。进一步分析显示：一期、二期梅毒拭子DNA阳性率（63.2%比 87.5%）差异无统计学意义（χ2 = 2.62，P > 0.05）；血清样本中，二期梅毒DNA阳性率高于一期梅毒（χ2 = 3.6，P = 0.06）；全血样本中，二期梅毒DNA阳性率高于其他各类型梅毒；神经梅毒耳垂末梢血阳性率与脑脊液比较，P = 0.06。隐性梅毒血清（RPR）滴度 ≥ 1∶8组的Tp DNA阳性率高于RPR ≤ 1∶4组。结论 NR?PCR方法检测梅毒患者不同样本中Tp DNA是可行的，不同类型梅毒、不同类型样本以及其血清RPR滴度水平都影响Tp DNA阳性率。

Abstract: Ye Xingdong, Gao Fangming, Cao Wenling, Lin Hongda, Ren Zefang Guangzhou Institute of Dermatology, Guangzhou 510095, China （Ye XD, Gao FM, Cao WL）; School of Public Health, Sun Yat?sen University, Guangzhou 510080, China （Lin HD, Ren ZF） Corresponding author: Ye Xingdong, Email: yxingdong@qq.com 【Abstract】 Objective To investigate the feasibility and prospects of nested real?time PCR （NR?PCR） technique for Treponema palladium （Tp） detection in various samples of different stages of syphilis from patients preliminarily diagnosed as syphilis. Methods Targeting the Tp polA gene, NR?PCR was performed to detect Tp DNA in various samples from the patients with various stages of syphilis at the first clinic visit, including skin tissue fluid swabs, serum, whole blood, cerebrospinal fluid （CSF） and earlobe blood. Data were analyzed with SPSS software version 13. Results A total of 368 clinical samples were collected from 200 patients with syphilis. With a detection limit of 2 Tp/ml, NR?PCR showed that the total positive rate for Tp DNA was 71.7% （264/368）. The Tp DNA positive rate was highest in earlobe blood samples （92.0%, 23/25）, followed by CSF samples （90.2%, 46/51）, skin tissue fluid swabs （74.3%, 26/35）, serum samples （66.9%, 99/148） and whole blood samples （64.2%, 70/109）. There was good agreement between NR?PCR results and serologic test results, with a consistency rate of 76.0% （152/200）. Furthermore, the Tp DNA positive rate did not differ between patients with primary （12/19） and secondary syphilis （14/16） in skin tissue fluid swabs （χ2 = 2.62, P > 0.05）, and was slightly but insignificantly higher in patients with secondary syphilis than those with primary syphilis in the serum samples （χ2 = 3.6, P = 0.06）. The Tp DNA positive rate of whole blood samples was also higher in patients with secondary syphilis than those with any other types of syphilis. Among patients with neurosyphilis, no significant difference was observed in the Tp DNA positive rate between earlobe blood samples and CSF samples （P = 0.06）. Among patients with latent syphilis, the Tp DNA positive rate was significantly higher in serum samples with an RPR titer of ≥ 1∶8 than those with an RPR titer of ≤ 1∶4. Conclusion NR?PCR is feasible for detecting Tp DNA in various kinds of samples, and the Tp DNA positive rate is influenced by stages of syphilis and types of samples, as well as RPR titers.

叶兴东高方铭曹文苓林宏达任泽舫. 巢式-实时PCR检测梅毒初诊患者不同样本中梅毒螺旋体靶DNA[J]. 中华皮肤科杂志, 2017,50(5):346-350. doi:

参考文献 16

［1］	Zobanikova, M, Mikolka, P, Cejkova, D, et al. Complete genome sequence of Treponema pallidum strain DAL⁃1［J］. Stand Genomic Sci,2012, 7（1）: 12⁃21. DOI: 10.4056/sigs.2615838.
［2］	李军, 郑和义. 梅毒检测研究进展［J］. 中国医学科学院学报,2012, 34（1）: 95⁃98. DOI: 10.3881/j.issn.1000⁃503X.2012.01. 018.
［3］	叶兴东, 林宏达, 戴向农, 等. 聚合酶链反应检测梅毒螺旋体的应用进展［J］. 国际皮肤性病学杂志, 2016, 42（1）: 15⁃18. DOI: 10.3760/cma.j.issn.1673⁃4173.2016.01.006.
［4］	中国疾病预防控制中心性病艾滋病预防控制中心. 梅毒、淋病、生殖器疱疹、生殖道沙眼衣原体感染诊疗指南（2014）［J］. 中华皮肤科杂志, 2014, 47（5）: 365⁃372. DOI: 10.3760/cma.j.issn.0412⁃4010.2014.05.022.
［5］	Liu, H, Rodes, B, Chen, CY, et al. New tests for syphilis: rational design of a PCR method for detection of Treponema pallidum in clinical specimens using unique regions of the DNA polymerase I gene［J］. J Clin Microbiol, 2001, 39（5）: 1941⁃1946. DOI: 10.1128/JCM.39.5. 1941⁃ 1946.2001.
［6］	Leslie DE, Azzato F, Karapanagiotidis T, et al. Development of a real⁃time PCR assay to detect Treponema pallidum in clinical specimens and assessment of the assay′s performance by comparison with serological testing［J］. J Clin Microbiol, 2007, 45（1）: 93⁃96. DOI: 10.1128/JCM. 01578⁃06.
［7］	Marra CM, Maxwell CL, Smith SL, et al. Cerebrospinal fluid abnormalities in patients with syphilis: association with clinical and laboratory features［J］. J Infect Dis, 2004, 189（3）: 369⁃376. DOI: 10.1086/381227.
［8］	曾铁兵, 吴移谋, 黄澍杰, 等. 巢式PCR扩增梅毒螺旋体polA及其临床应用的研究［J］. 中国皮肤性病学杂志, 2004, 18（2）: 15⁃17. DOI: 1001 ⁃7089（2004）02⁃0074⁃03.
［9］	Grange PA, Gressier L, Dion, PL, et al. Evaluation of a PCR test for detection of treponema pallidum in swabs and blood［J］. J Clin Microbiol, 2012, 50（3）: 546⁃552. DOI: 10.1128/JCM.00702 ⁃11.
［10］	Chi KH, Danavall D, Taleo F, et al. Molecular Differentiation of Treponema pallidum subspecies in skin ulceration clinically suspected as yaws in vanuatu using real⁃time multiplex PCR and serological methods［J］. Am J Trop Med Hyg, 2015, 92（1）: 134⁃138. DOI: 10.4269/ajtmh.14⁃0459.
［11］	Gayet⁃Ageron A, Ninet B, Toutous⁃Trellu L, et al. Assessment of a real⁃time PCR test to diagnose syphilis from diverse biological samples［J］. Sex Transm Infect, 2009, 85（4）: 264⁃269. DOI: 10.1136/sti.2008.034314.
［12］	Sutton MY, Liu H, Steiner B, et al. Molecular subtyping of Treponema pallidum in an Arizona County with increasing syphilis morbidity: use of specimens from ulcers and blood［J］. J Infect Dis, 2001, 183（11）: 1601⁃1606. DOI: 10.1086/320698.
［13］	Castro R, Prieto E, Aguas MJ, et al. Detection of Treponema pallidum sp pallidum DNA in latent syphilis［J］. Int J STD AIDS, 2007, 18（12）: 842⁃845. DOI: 10.1258/095646207782716901.
［14］	Flasarova M, Pospisilova P, Mikalova L, et al. Sequencing⁃based molecular typing of treponema pallidum strains in the Czech Republic: all identified genotypes are related to the sequence of the SS14 strain［J］. Acta Derm Venereol, 2012, 92（6）: 669⁃674. DOI: 10.2340/00015555⁃1335.
［15］	Gupte S, Daly C, Agarwal V, et al. Introduction of rapid tests for large⁃scale syphilis screening among female, male, and trans⁃gender sex workers in Mumbai, India［J］. Sex Transm Dis, 2011, 38（6）: 499⁃502. DOI: 10.1097/OLQ.0b013e318205e45d.
［16］	De Santis M, De Luca C, Mappa I, et al. Syphilis infection during pregnancy: fetal risks and clinical management［J］. Infect Dis Obstet Gynecol, 2012, 2012: 430585. DOI: 10.1155/2012/430585.