Study On The Detection Of Perinatal Chlamydia Trachomatis Infections By Urine Gap-LCR-ELISA And Genotype

Part I Detection of Chlamydia Trachomatis by plasmid Gap-LCR-ELISA【Objective】To construct a highly sensitive and specific plasmid Gap-LCR-ELISA method to detect Chlamydia Trachomatis (CT).【Methods】Four labelled oligonucleotides probes derived from CT cryptic plasmid were used for Gap-LCR. With the quantified CT standard serovars B-TW-5 (2.5×105EB/ml), a series of tests were performed to optimize Gap-LCR amplification conditions, which were chosen according to the most abundant optic density (OD) of the amplified fragment after PAGE. Gap-LCR was conducted in six CT serovars as well as in other bacteria frequently causing genitourinary tract infections. PCR was performed with the same plasmid primers as the methodology control. All the amplified products were detected by ELISA to assess the sensitivity and specificity of plasmid Gap-LCR-ELISA.【Results】①The optimized conditions of Gap-LCR reaction was as follows: 5μl DNA sample was used in a total 20μl reaction volume consisting of 2μl of 10×LCR buffer, 0.5μl of 10mM dGTP, 0.5μl of 25pmol/μl each probe, 5U of Tsc DNA ligase thermostable, 3U of Taq DNA Polymerase. This was first pre-denatured at 94℃ for 5min, then 40 cycles of denaturing at 94℃ for 30sec, followed by annealing and ligation at 58℃ for 120sec. ②The extracted DNA from six CT serovars all presented positive results, while the C psittaci D34 and other common urogenital pathogens had negative results. ③The detectable level of CT serovar B-TW-5 was greater than 2.5EB, whereas PCR-ELISA (using the same genetically conservative primers) could only identify those with more than 25 EB, which showed that the detectable level of CT using Gap-LCR-ELISA was ten times more sensitive than that of PCR-ELISA.【Conclusions】Plasmid Gap-LCR-ELISA possesses a very high specificity for CT, without false positives from other chlamydiae species or from other common urogenital pathogens. In addition, this technique has a high sensitivity, able to detect CT serovars at 2.5EB. Its ability is comparable to Abbott IMxTm System (U.S.A), so it is a very promising and efficient method which may be used to identify CT infections in the urogenital tract of asymptomatic patients in grass-root medical units.