Multiplex PCR Assay for Chlamydia Trachomatis & Neisseria Gonorrhoeae
Introduction
Chlamydia trachomatis and Neisseria gonorrhoeae are the two most common sexually transmitted bacterial pathogens. They are responsible for a wide variety of acute clinical conditions and for important long-term consequences that adversely affect women's reproductive health. Chlamydia trachomatis and Neisseria gonorrhoeae infections are now recognized as the leading cause of sexually transmitted diseases (STDs).
Conventional Methods
Conventional diagnosis of N. gonorrhoeae infection requires isolation on selection media or observation of gram-negative diplococci in Gram smears of genital discharge, or urethral or cervical swabs. Although culture is relatively inexpensive and highly sensitive, it is logistically complicated. A pelvic examination is required for women, and insertion of a urethral swab is required for men. Diagnosis of C. trachomatis infection is frequently based on isolation in tissue culture. This procedure requires careful specimen collection and stringent transport conditions and requires 48 to 72 hrs to perform.
Principles of the Procedure
The CT/NG Test is a multiplex assay that permits the simultaneous amplification of C. trachomatis target DNA, N. gonorrhoeae target DNA and internal control DNA. The Master Mix reagent contains biotinylated primer pair specific for C. trachomatis, N. gonorrhoeae and internal control.
Sample Preparation
Epithelial cells, leukocytes and associated C. trachomatis & N. gonorrhoeae cells, collect on swabs or pelleted from urine are treated with detergent solution to release both the chlamydial DNA and the neisseria DNA.
PCR Amplification
The processed specimens are added to the amplification mixture in reaction tubes in which PCR takes place. The process is repeated for a number of cycles, each cycle effectively doubling the amount of amplicons.
Detection
Detection uses the hybridization reaction of amplicon to the target specific probe, which is followed by the addition of AV-HRP to detect the CT/NG.
Salient Features
The AMPLICOR® CT/NG Test offers
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Clearance by the FDA for commercial marketing |
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High sensitivity without a loss of specificity |
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Increased sensitivity over traditional culture or DNA direct probe tests therefore, more positive specimens can be detected |
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An ideal screening application (high sensitivity in low prevalence populations, including asymptomatic and symptomatic patients) |
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Detection of two of the most common STD pathogens in a single test from one single specimen processing |
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Optional co-amplified internal control enabling inhibition control |
References |
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Gomes JP, Ferreira MA, De Sa AB, Catry MA.2001. Pooling urine samples for PCR screening of C. trachomatis urogenital infection in women. Sex Transm Infect. Feb;77(1):76-7 |
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Loeffelholz, M.J., Lewinski, C.A., Silver, S.R. 1992. Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. Journal of Clinical Microbiology 30:2847-2851 |
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Miyada, C.G. and Born, T.L. 1991. A DNA sequence for the discrimination of Neisseria gonorrhoeae from other Neisseria species. Molecular and Cellular Probes 5:327-335 |
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