双引物聚合酶链反应同时快速检测梅毒螺旋体和单纯疱疹病毒Ⅱ型

SIMULTANEOUS EXAMINATION OF HERPES SIMPLEX VIRUS Ⅱ AND TREPONEMA PALLIDUM BY DOUBLE-PRIMER PCR

目的建立双引物聚合酶链反应(PCR)用于同时快速检测梅毒螺旋体和单纯疱疹病毒Ⅱ型。方法基于单纯疱疹病毒II型(HSV-2)的DNA聚合酶基因以及梅毒螺旋体47KD膜蛋白基因序列,自行设计2对特异性寡核苷酸引物,建立了同时检测上述2种病原体的双引物PCR。结果各特异性引物仅扩增相应的病原体DNA,即单纯疱疹病毒Ⅱ型扩增产物为202bp以及梅毒螺旋体扩增产物为252bp。其他11种生殖道常见微生物及正常人基因组DNA不被扩增。检测了51例临床标本,梅毒螺旋体暗视野显微镜检查阳性检出率为15.7%,双引物PCR阳性检出率为19.6%;二种方法检测结果间比较差异均无统计学意义(P>0.05)。HSV阳性培养率23.6%,双引物PCR阳性率47.1%,双引物PCR阳性检出率显著高于HSV培养且差异有统计学意义(P=0.019);上述临床标本分别进行了2种病原体的单引物PCR,与双引物PCR对照结果完全一致。结论我们建立的双引物PCR一次可同时检测2种病原体,且有快速、敏感、特异以及简便的特点。

Objective To approach double- primer PCR assay for simultaneous detection of Herpes simplex virus Ⅱ （HSV - 2） and Treponema pallidum. Methods According to specific gene sequence of herpes simplex virus Ⅱ DNA Polymerse gene and treponema pallidum 47 kd gene, 2 sets of specific primers were designed and a double - primer PCR assay were developed to detect 2 agents in one test. Results The target DNAs of 2 agents were specifically arhplified by theirs specific primers. The DNA fragment length of amplifying HSV - 2 was 202 bp, amplifying treponema pallidum was 252 bp; The DNA of other 11 common microbes of genital tract and human gene were not amplified by 2 sets of primers. 51 clinical samples were detected by darkfield microscopy and a doubleprimer PCR, the positive rates were 15.7% and 19.6%, respectively. And there was no statistically significant different between darkfield microscopy and a double- primer PCR （P〉0.05）. Detected by HSV culture and a double - primer PCR, the positive rates were 23.6% and 47. 1% respectively. The sensitivity of Hsv detected by PCR and a double- primer PCR was significantly higher than that of culture, and there was significant different between HSV culture and PCR and a double-primer PCR （P = 0. 019）. All specimens were tested for HSV and Treponema pallidum by single- primer PCR respectively, the results were consistent to double- primer PCR. Conclusion Primary study indicates that the double- primer PCR assay can simultaneously, specifically, rapidly, sensitively detect 2 agents.