半巢式聚合酶链反应-微孔板杂交法检测沙眼衣原体

目的：应用半巢式聚合酶链反应－微孔板杂交法（半巢式PCR－MPH）检测泌尿生殖道沙眼衣原体（Ct)。方法：分别用质粒和主要外膜蛋白基因引物进行扩增，将下游引物用生物素标记，经半巢式PCR扩增Ct主要外膜蛋白基因后，扩增产物被包被有链霉亲和素的微孔板捕获，经碱变性后与标有荧光素的特异性探针进行杂交，然后通过辣根酶标记的抗荧光素抗体与杂交分子反应，经过酶底物显色，通过测定吸光度来判断结果。结果：主要外膜蛋白基因引物半巢式PCR较质粒引物PCR更敏感，比半巢式PCR产物酶切后电泳法高10倍，半巢式PCR－MPH法特异性强，该法重复法好，批内CV值＜10％，批间CV值＜15％，该法在包被浓度为4mg/L，产物1：20稀释，与微孔板结合20min后加入10pmol/mL探针杂交30min,用1：3000稀释的酶显色条件下有最佳结果，结论：该法操作简便，敏感性和特异性均高，无放射性和EB染料污染，适于临床样本的常规检测。

Application of Semi-nested Polymerase Chain Reaction in Detection of Chlamydia Trachomatis

Objective:To investigate the value of semi-nested PCR-MPH in diagnosis of Chlamysia trachomatis (Ct) infection in the urogenital tract. Methods:Plasmid and major outer membrane protein gene(ompl) primer were used to amplify Ct DNA. The downprimer of MOMP gene was labeled by biotin,and the omplgene amplification product was captured on streptavidin-coated microplates and denatured by NaOH. Then the product was quantified by hybridization with a fluorescein-coated microplates and denatured by probe. After revelation with an anti-fluorescein horse-radish peroxidase coupled antibody (anti-FITC-POD), the mount of hybridized probe was determined by optical reading. Results:Semi-nested PCR-MPH method could detect Ct DNA with high sensitivity, specificity and accuracy. Conclusion:The semi-nested polymerase chain reaction is useful for routine Ct DNA detection of clinical specimens.