半巢式聚合酶链反应-微孔板杂交法检测沙眼衣原体

目的 建立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

Chlamydia trachomatis detected by semi-nested polymerase chain reaction

Objective To establish a semi nested PCR MPH method for sensitive, specific and fast diagnosis of Chlamysia trachomatis (Ct) infected in the urogenital tract.Methods We used plasmid and major outer membrane protein gene (omp1) primer to amplify Ct DNA separately. The down primer of MOMP gene was labeled by biotin. The amplification product was captured on streptavidin coated microplates and denatured by NaOH. Then the products were quantified by hybridization with a fluorescein labeled internal oligonucleotide probe. After revelation with an anti fluorescein horse radish peroxidase coupled antibody (anti FITC POD), the amount of hybridized probe was determined by optical reading.Results The plasmid primer was more sensitive than omp1 primer. Semi nested PCR MPH method developed by us could detect Ct DNA with high sensitivity, specificity, and accuracy.Conclusion The method is of potential use for routine Ct DNA detection of clinical specimens.