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

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

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。

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.