淋病奈瑟菌、沙眼衣原体、细小脲原体多重荧光定量PCR检测方法的建立及临床应用

Development and clinical application of multiple fluorescent quantitative PCR methods for simultaneous detection of Neisseria agonorrhoeae, Chlamydia trachomatis and Ureaplasma parvum

目的建立淋病奈瑟菌（Neisseriaagonorrhoeae，NG）、沙眼衣原体（Chlamydiatrachomatis，CT）、细小脲原体（Ureaplasmaparvum，uP）多重荧光定量PCR（fluorescentquantitativePCR，FQ—PCR）检测方法。方法选择NG保守基因porA、解脲支原体（Ureaplasmaurealytieum，uu）保守基因UPq及cT保守基因￡rp作为目标检测基因，设计引物及TaqMan探针，制备重组质粒标准品，绘制标准曲线，建立多重VQ．PCR检测方法，并验证其敏感性、特异性及重复性。用建立的方法对203份泌尿生殖道感染NG、CT、UP的临床样本进行同步检测，并与单重VQ—PCR法、常规PCR法及基因测序结果进行比较。结果重组质粒标准品经双酶切及测序鉴定，证明构建正确；建立的标准曲线起始DNA拷贝数与0值之间的线性关系良好，相关系数为0．998～1．000；该方法检测NG、CT和uP的灵敏度均可达10：copies／ml，检测范围可达10。～10。copies／ml，相关系数分别为1．000、0．999和0．995；不与生殖道其他菌群如阴道霉菌、百日咳杆菌、流感嗜血杆菌、人型支原体、单纯疱疹病毒I型、Ⅱ型及人乳头瘤病毒（16／18型）发生交叉反应；检测的203份样本中，多重VQ—PCR检测NG、CT、UP的阳性检出率分别为34．48％、30．54％、18．71％，与单重VQ．PCR法、基因测序法比较，差异无统计学意义（P〉0．05），3种方法阳性检出率均高于常规PCR法；与基因测序法相比，多重FQ—PCR法灵敏度为100％，3种病原体检测特异性均高于98．50％。结论已成功建立NG、CT、UP多重VQ．PCR检测方法，该法检测时间短，特异性强，灵敏性高，适合于临床快速诊断；

Objective To develop a multiple fluorescent quantitative PCR（FQ-PCR） method for simuhaneous detection of Neisseria agonorrhoeae （NG）, Chlamydia trachomatis （CT） and Ureaplasmaparvum （UP）. Methods The conserved genes porA of NG, ure of Ureaplasma urealyticum （UU） and trp of CT were chosen as targets for design of primers and TaqMan probe. The standard recombinant plasmid was prepared, based on which a standard curve was plotted, and a multiplex FQ-PCR method was developed and verified for sensitivity, specificity and reproducibility. A total of 203 clinical samples were simultaneously detected for NG, CT and UP by the developed method, and the results were compared with those by simplex FQ-PCR, routine PCR and gene sequencing. Results Standard recombinant plasmid was constructed correctly as proved by restriction analysis and sequencing. The standard curve showed good linear relationship between starting DNA copy number and Ct value, with a correlation coefficient of 0. 998 - 1. 000. The sensitivities of the developed method for NG, CT and UP were 102 copies/ml, while the detection ranges were 109 - 102 copies/ml, with correlation coefficients of 1. 000, 0. 999 and 0. 995 respectively. No cross reactions with other microbial floras, such as vaginal mould, Bordetella pertussis, Haemophilus influenzae, Mycoplcasma hominis, herpes simplex virus types I and II and human papillomavirus （16/18）, were observed. In the 203 clinical samples, the positive rates ofNG, CT and UP were 34. 48% （70/203）, 30. 54% （62/203） and 18. 71% （38/203） respectively by the developed multiple FQ-PCR, which showed no significant difference with those by simplex FQ-PCR and gene sequencing （P 〉 O. 05 ）, and were higher than those by routine PCR. Compared with gene sequencing, the sensitivity of multiplex FQ-PCR was 100%, while the specificities for all the three pathogens were more than 98. 50%. Conclusion A multiplex FQ-PCR for simultaneous detection of NG, CT and UP was successfully developed, which was time-saving, specific, sensitive, and suitable for rapid diagnosis in clinic.