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Gene typing of Chlamydia trachomatis by polymerase chain reaction and restriction endonuclease digestion
1Division of Infectious Diseases, Johns Hopkins University Hospital, Baltimore, MD 21205.
Abstract:
A portion of the major outer membrane protein (MOMP) gene from 15 Chlamydia trachomatis serovars was amplified by polymerase chain reaction (PCR) and the product was analyzed by restriction fragment length polymorphism (RFLP). A set of primers was used to amplify an 871 base pair gene fragment encompassing the 4 hypervariable regions of MOMP. AluI digestion of the product gave distinctive patterns for the 15 serovars as demonstrated on silver-stained polyacrylamide gels. A triple digest with EcoRI, HinfI, and HpaII allowed improved discrimination between closely related serovars (C, H, I, J, L3). PCR and RFLP were used to type 50 wild-type clinical isolates and results were compared to results of the solid-phase enzyme immunoassay typing method. These isolates represented the most prevalent genital serovars (D, E, F, K, I and J) in the local sexually transmitted diseases clinic population. For specimens containing 1 serovar, the results of the two methods were similar for 42 samples and discordant for 1 sample. In addition, two samples showed evidence of mixed infection with two serovars as identified by both methods. Five additional specimens contained two serovars, as shown by one or both methods. In all five such specimens, the two typing methods agreed on at least one of the two serovars. For both single and multiple serovar specimens, there was concordance between the two typing methods for 16/17 E serovars, 8/9 D serovars, 8/8 F serovars, 7/7 I serovars, 7/7 J serovars, 5/8 K serovars, and 0/2 G serovars.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
This study introduces a new method using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) to accurately identify Chlamydia trachomatis serovars. This technique improves the detection of single and mixed infections, crucial for sexually transmitted disease diagnostics.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Chlamydia trachomatis serovar identification is critical for epidemiological surveillance and clinical management of sexually transmitted infections.
- Current typing methods may have limitations in discriminating between closely related serovars or detecting mixed infections.
Purpose of the Study:
- To develop and validate a novel typing method for Chlamydia trachomatis serovars using PCR and RFLP.
- To compare the performance of the new PCR-RFLP method with existing solid-phase enzyme immunoassay typing.
Main Methods:
- Amplification of a major outer membrane protein (MOMP) gene fragment from 15 Chlamydia trachomatis serovars using PCR.
- Analysis of the PCR product by restriction fragment length polymorphism (RFLP) using AluI and a triple digest (EcoRI, HinfI, HpaII).
- Typing of 50 wild-type clinical isolates and comparison with solid-phase enzyme immunoassay results.
Main Results:
- Distinctive RFLP patterns were observed for the 15 serovars using AluI digestion.
- A triple digest improved discrimination between closely related serovars.
- High concordance was found between PCR-RFLP and enzyme immunoassay for single and mixed serovar infections, with notable agreement for prevalent serovars like E, D, F, I, and J.
Conclusions:
- PCR-RFLP is a reliable and effective method for typing Chlamydia trachomatis serovars.
- This method offers improved discrimination and can accurately identify single and mixed infections.
- The findings support the utility of PCR-RFLP for clinical diagnostics and epidemiological studies of Chlamydia trachomatis.