c. trachoamtis detection and genotypings assay
TRANSCRIPT
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Development of a C.trachomatis amplification, detection and genotypings assay
By Koen Quint
DDL diagnostics laboratory
Leiden University
Email:[email protected]
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Outline
• Introduction
• Ct amplification assay
• Ct detection assay
• Ct genotypings assay
• Conclusions
• Ct cofactor study
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Introduction
• In the ’70 culture systems were the golden standard.
• Serology was also used to distinguish between acute and chronic infections.
• EIA assays were a first quick alternative for culture.
• DNA-probes and Nucleid Acid Amplification Tests (NAAT) have a high sensitivity relative to culture.
• The second generation NAATs are developed to improve the specificity (cross-reaction, contamination etc.) and sensitivity (the Swedish variant).
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The different serovars of C. trachomatis display diverse biological activity.
• Serovar A, B/Ba and C are commonly associated with an ocular disease, trachoma. Serovars B and C are rarely detected in the urogenital tract.
• Serovars D/Da, E, F, G/Ga, H, I/Ia, J and K are common in the urogenital tract and can sometimes be detected in the respiratory tract or eye of newborns.
• Serovars L1, L2/L2a, and L3 are mainly detected in the inguinal lymph nodes and the rectum, and may cause lymphogranuloma venereum.
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Development of a Ct-Amplification, Ct-Detection and Ct-Genotyping assay
• The Ct-Amplification assay comprises a Ct-multiplex-broad-spectrum PCR primer mix with multiple forward and reverse primers.
• The Ct-Detection assay comprises a DNA enzyme immuno assay (DEIA) with a mix of conserved probes.
• The Ct-Genotyping assay is based on the reverse hybridisation methodology allowing the simultaneous identification of multiple C. trachomatis serovars in a single hybridization step.
Produced :Labo Biomedical Products BV, Rijswijk, The Netherlands
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Ct amplification step
Ct detection step
Ct genotyping step
Cervical scrape DNA isolations
CT-negative CT-positive
Detection in microtiterplate hybridization assay
CT genotypes
Algorithm in Detection and Genotyping of Ct
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0
Ct amplification step with multiplex broad-spectrum PCR
CS1
VS1 VS2 VS3 VS4
CS2 CS3 CS4 CS5
950 bp
Bacterial chromosome, ompI
160 bp
Cryptic plasmid
89 bp
Probe region
Probe region
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Phylogenetic tree of 160 bp amplicon of C. trachomatis
Group B
Group C
Interm. group
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Ct Detection Assay
• DNA enzyme immuno assay for screening
• Mix of conserved probes (based on the cryptic plasmid amplicon as well as omp1 amplicon)
• Within 5 hours for 93 results (+3 controls)
• More sensitive then an agarose gel
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Comparison Ct-Dt assay with Cobas Taqman (Roche)
Quint K et al, J. Mol. Diagn. 2007 vol. 9 p.631
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Comparison Ct-Dt assay with Hybrid Capture2 (Digene)
Quint K et al. J. Clin. Microbiol. 2007 vol 45 p3986
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Strip
DNA-probe
BiotinStreptavidin
Alkaline phosphatase Substrate
Purple precipitate
PCR-amplified target
Principle of reverse hybridization analysis The Ct-Genotyping assay
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Outline and specificity of the Ct-genotyping assay
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Clinical examples of multiple infections
Quint K et al, J. Mol. Diagn. 2007 vol. 9 p.631
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Serovar distribution for 4 different countries
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Conclusion
• The amplification assay will generate two amplicons (one based on cryptic plasmid and one based on the omp1 gene)
• The Ct-detection test has the same sensitivity as the COBAS TaqMan and detects significant more Ct infections compared with the HC2 test.
• The Ct- genotyping test is specific for all available (in the genebank) serovars. Multiple infections within the same serogroup need to be sequenced
• The Ct-genotyping test is a quick method for serovar distribution studies.