裂解酶片段长度多态性分析在母婴垂直传播沙眼衣原体主要外膜蛋白基因分型中的应用

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 ;了解重庆地区近年来临产孕妇Ct感染和母婴垂直传播状况及常见基因型。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 30 0对 6 0 5份 ,采用Ctomp1套式聚合酶链反应 (omp1 nPCR)检测Ct;用建立的CFLP方法对临床Ct分离株进行分型。结果　孕妇宫颈Ct检出率 11% (33/ 30 0 ) ,母婴传播率 2 4 2 % (8/ 33)。生后 2 4h内和 5～ 10d采集Ct阳性母亲所生新生儿鼻咽拭子标本Ct检出率分别为 3 0 % (1/ 33)和 38 9% (7/ 18) ,χc2 =8 79,P <0 0 1。剖宫产和阴道分娩母婴传播率分别为 8 3% (2 / 2 4 )和 6 6 7% (6 / 9) ,χc2 =9 16 ,P <0 0 1。Ct阳性和阴性孕妇胎膜早破发生率分别为 30 3% (10 / 33)和 13 5 % (36 / 2 6 7) ,χ2 =6 4 0 ,P <0 0 5。 8对Ct阳性母婴配对标本CFLP图谱呈 4类 ,经测序证实分别为E、F、H、D型 (各 3、2、2、1对 ) ,各占 37 5 %、2 5 0 %、2 5 0 %和 12 5 % ,且每对母子CFLP图谱完全一致。结论　本研究结果在一定意义上反映了重庆地区近年来临产孕妇Ct感染和母婴垂直传播状况及常见基因型 ;上游引物 5′末端地高辛标记Ctomp1CFLP分型方法灵敏度

Genotyping of major outer membrane protein gene of Chlamydia trachomatis by cleavase fragment length polymorphism analysis

OBJECTIVE: To establish a methed of cleavage fragment length polymorphism (CFLP) analysis with a primer labeled at the 5'-end with digoxigenin for genotyping of Chlamydia trachomatis (Ct). The methods for detection of Ct by major outer membrane protein (MOMP) gene (ompl) with nested polymerase chain reaction (ompl-nPCR) were studied. The incidence of Ct infection in pregnant women, the common genotypes and vertical transmission rate of Ct in Chongqing area during the past one year was also investigated. METHODS: The samples were taken from cervical scrapes of parturient women and nasopharygeal swabs of their neonates from April 2003 to Feb. 2004 in Chongqing Women and Children's Health Care Institute. Totally 300 pairs (605 specimens) were detected by using ompl-nPCR, ompl-PCR (inside pair of primers was used directly) and plasmid-PCR. The results were judged by the modified gold standard (MGS). The ompl-nPCR amplified DNA was purified by recovery of DNA from agarose gel electroelution into dialysis bags. The DNA amplified from ompl-nPCR was sequenced by ABI PRISM 377 DNA sequencer. CFLP assay with a primer labeled at the 5'-end with digoxigenin was created for genotyping of Ct, and was primarily applied. RESULTS: The minimum detectable levels of ompl-nPCR and ompl-PCR corresponded to 2.5 elementary body (EB) and 25 EB, respectively. The sensitivity of ompl-nPCR was 10 times that of ompl-PCR. 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). The rate of Ct isolated from nasopharyngeal swabs 5 - 10 days after birth was 38.9% (7/18), which was significantly greater than that [3.0% (1/33)] detected within 24 hours after birth (chi(c)(2) = 8.79, P < 0.01). Of the 33 Ct-positive samples from pregnant women, 9 had vaginal delivery and 24 had caesarean section. The vertical transmission rates in vaginal delivery group and caesarean section group were 66.7% (6/9) and 8.3% (2/24), respectively (chi(c)(2) = 9.16, P < 0.01). Incidence of premature rupture of membrane in Ct-positive group was 30.3% (10/33), which was greater than that of Ct-negative groups (13.5%, 36/267, chi(2) = 6.40, P < 0.05). Four different patterns were observed in the 16 Ct-positive samples from 8 pregnant women and 8 matched maternal-infants by using CFLP, which were confirmed by DNA sequencing later. They were type E (3 pairs), type F (2 pairs), type H (2 pairs) and type D (1 pair). Each pair of matched maternal-infantile samples presented identical CFLP pattern. CONCLUSIONS: This study revealed the infection rate of Ct in pregnant women, vertical transmission rate of Ct and the common genotypes of Ct in Chongqing Women and Children's Health Care Institute. The CFLP assay by using a primer labeled at the 5'-end with digoxigenin was first used for genotyping of Ct. The assay showed a good sensitivity and reproducibility, no radioactive contamination, and is simple. Therefore the assay is a potential new method for Ct genotyping.