【摘要】 目的建立沙眼衣原体(Ct)主要外膜蛋白基因(omp1)上游引物5′末端地高辛标记裂解酶片段长度多态性(CFLP)分型方法,并对Ct临床分离株进行分型。方法取临产孕妇宫颈刮片及其新生儿鼻咽拭子组成母婴配对标本300对605份,用套式聚合酶链反应扩增Ctomp1第四可变区;用建立的CFLP方法对其进行基因分型。结果孕妇宫颈Ct检出率11%(33/300),母婴传播率24.2%(8/33);8对Ct阳性母婴配对标本CFLP图谱呈四类,经测序证实分别为E、F、H、D型(各3、2、2、1对),各占37.5%、25%、25%和12.5%,且每对母子CFLP图谱完全一致;同一标本不同批次和相同基因型不同标本的CFLP图谱一致。结论上游引物5′末端地高辛标记Ctomp1CFLP分型方法灵敏度高、重复性好、操作简便、快速、费用相对低廉,无放射污染,是一种极具潜力的Ct基因分型方法。更多还原

【Abstract】 Objective To establish a method of cleavase fragment length polymorphism (CFLP) analysis for genotyping of Chlamydia trachomatis (Ct).Methods The samples were taken from cervical scrapes of parturient women and nasopharygeal swabs of their neonates. Total 300 pairs (605 specimen) were detected by nested polymerase chain reaction amplifying major outer membrane protein gene (omp1). CFLP assay labeled at the 5′-end of the forward primer with digoxigenin was created for genotyping of Ct and primarily applied.Results The positive rate of Ct in the samples from the pregnant women was 11% (33/300). The vertical transmission rate of Ct from mothers to their infants was 24.2% (8/33). Four kinds of different CFLP patterns were observed in the 16 Ct-positive samples from 8 pregnant women and 8 matched maternal-infants, which were confirmed by DNA sequencing. They were type E (3 pairs), type F (2 pairs), type H (2 pairs) and type D (1 pair), accounting for 37.5%, 25%, 25%, 12.5% respectively. 8% denaturing polyacrylamide gel, incubation time of 10 minutes and initial ramping temperature of 40℃ were examined to reveal conditions yielding an optimal CFLP pattern. Each pair of matched maternal-infantile samples presented identical CFLP pattern. CFLP analysis in different batches reproduced identical pattern.Conclusions The CFLP assay labeled at the 5′-end of the forward primer with digoxigenin was first successfully used for genotyping of Ct. With good sensitivity, reproducibility, no radioactive contamination and simple, it was a potential new method for Ct genotyping.更多还原