Development of a C.trachomatis amplification, detection and genotypings assay By Koen Quint DDL diagnostics laboratory Leiden University Email:[email protected]
Jul 14, 2015
Development of a C.trachomatis amplification, detection and genotypings assay
By Koen Quint
DDL diagnostics laboratory
Leiden University
Email:[email protected]
Outline
• Introduction
• Ct amplification assay
• Ct detection assay
• Ct genotypings assay
• Conclusions
• Ct cofactor study
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).
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.
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
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
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
Phylogenetic tree of 160 bp amplicon of C. trachomatis
Group B
Group C
Interm. group
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
Comparison Ct-Dt assay with Cobas Taqman (Roche)
Quint K et al, J. Mol. Diagn. 2007 vol. 9 p.631
Comparison Ct-Dt assay with Hybrid Capture2 (Digene)
Quint K et al. J. Clin. Microbiol. 2007 vol 45 p3986
Strip
DNA-probe
BiotinStreptavidin
Alkaline phosphatase Substrate
Purple precipitate
PCR-amplified target
Principle of reverse hybridization analysis The Ct-Genotyping assay
Outline and specificity of the Ct-genotyping assay
Clinical examples of multiple infections
Quint K et al, J. Mol. Diagn. 2007 vol. 9 p.631
Serovar distribution for 4 different countries
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.