| Objective To establish the methed of cleavase fragment length polymorphism (CFLP) analysis labeled at the 5′-end of one primer with digoxigenin for genotyping of chlamydia trachomatis (Ct). The methods for detection of Ct by major outer membrane protein (MOMP)gene (ompl) nested polymerase chain reaction (ompl-nPCR), DNA purification and DNA sequencing were studied and compared. 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 were also investigated. Methods The samples were taken from cervical scrapes of parturient women and nasopharygeal swabs of their neonates from Apr. 2003 to Feb. 2004 in Chongqing Women and Children's Health Care Institute. Total 300 pairs (605 specimen) were detected by 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 DNA was purificated by recovery of DNA from agarose gels electroelution into dialysis bags. The DNA amplified from ompl-nPCR was sequenced by ABI PRISM 377 DNA sequencer. CFLP assay labeled at the 5′-end of one primer with digoxigenin was created for genotyping of Ct, and was primarily applied.Results The minimum detectable levels of ompl-nPCR and ompl-PCR were 2.5 EB and 25 EB, respectively. The sensitivity of ompl-nPCR was 10 times of ompl-PCR. The sensitivity rate of ompl-nPCR and ompl-PCR were 100%(33/33)and 87.9%(29/33), respectively. The specificities were 99.6%(266/267)and 99.3%(265/267), respectively. 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 nasopharyngeal swabs taken in 24 hour and 5~10 days after birth were detected. The positive detected rate of Ct in the later samples was 38.9% (7/18), which was significantly greater than that of 3.0% (1/33) in the samples of first 24 hours (χ2=5.73,p<0.025). 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. The rate 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, χ2=4.2,p<0.05).Four kinds of 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 are 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 These 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 labeled at the 5′-end of one primer with digoxigenin was first used for genotyping of Ct. It showed the good sensitivity and reproducibility, no radioactive contamination, and is simple. It was a potential new method for Ct genotyping. |
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