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IPNC Juillet 2006

Real-Time PCR Applications:. Detection of chromosomally mediated penicillin-resistant Neisseria gonorrhoeae Quantitative expression of mRNA encoding pro-inflammatory and immunomodulatory cytokines in hamsters infected with a virulent variant of Leptospira. IPNC Juillet 2006.

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IPNC Juillet 2006

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  1. Real-Time PCR Applications: • Detection of chromosomally mediated penicillin-resistant Neisseria gonorrhoeae • Quantitative expression of mRNA encoding pro-inflammatory and immunomodulatory cytokines in hamsters infected with a virulent variant of Leptospira IPNC Juillet 2006

  2. Neisseria gonorrhoeae the etiologic agent for the sexually transmitted infection gonorrhoea • Second most common bacterial STD in France as in US • Around 75% of all reported cases in 15-29 year old age group • High prevalences reported from non-genital sites among MSM (oropharynx & rectum) • Usually symptomatic in males, often asymptomatic in women • Can cause PID, infertility, ectopic pregnancy, and complications in pregnancy in women Présentation des activités de l’Institut Pasteur

  3. Treatment Depending of the national guidelines • 1940s = Penicillin effective treatment • 1970s = Penicillin resistance develops secondary to acquisition of plasmid for penicillinase • Since 1972, CDC has recommended treatment with third generation cephalosporin or fluoroquinolones (SD) • Increase in chromosomally acquired multi-resistance to antibiotics • In New Caledonia, N.gonorrhoeae were susceptible to penicillin • Benzylpenicillin, metronidazole and azithromycin Présentation des activités de l’Institut Pasteur

  4. Mechanisms of antibiotic resistance • Alteration of the target site • Reduction of the affinity for the antibiotic • Access of the antibiotic to the target site • Reduced permeability of the cell envelope • Active export of antibiotics • Destruction of the antibiotic Présentation des activités de l’Institut Pasteur

  5. b-lactams action mode Antibiotics Penicillin binding proteins Présentation des activités de l’Institut Pasteur

  6. Mechanisms of penicillin resistance in N. Gonorrhoeae (1) Plasmid mediated b-lactamases Chromosomally- mediated alterations • Altered penicillin binding proteins • Porins • Efflux pumps Acquired, Transferable (e.g. TEM) is of considerable concern Présentation des activités de l’Institut Pasteur

  7. Mechanisms of penicillin resistance in N.gonorrhoeae (2) Présentation des activités de l’Institut Pasteur

  8. Real-Time Multiplex PCR Assay • Detection of NG from clinical samples within 2hrs • Combined detection of penA and ponA genotypes • ponA mutation = T to C substitution (CMI ≥ 1 mg/ml) • penA mutation = additional aspartic acid (Asp-345A) codon (CMI ≥ 0.03 mg/ml) • Correlation with Phenotypes obtained by E-test • Status of N.gonorrhoeae strains in New Caledonia • LightCycler: additional advantage Présentation des activités de l’Institut Pasteur

  9. Oligonucleotidesequences Design with Probe design software 2.0(Roche Diagnostics) aGenBank accession number; b LC-R705, LightCycler red 705; c LC-R640, LightCycler red 640 Présentation des activités de l’Institut Pasteur

  10. FRET based-system for the detection of ponA mutation (1) T C or 881 bp 3’ 5’ ponA-F ponA-P1 ponA-P2 FL LC705 5’ 3’ GGCGTTGGGCGGTGGT AAGAGCCGTTGCTGCA(PO4) A 814 1232 D 1265 hυ ponA-R FRET 5’ 1679 5’ 3’ Reverse Primer Forward Primer Anchor Probe Sensor Probe or Detection Probe Présentation des activités de l’Institut Pasteur

  11. Tm and mutation detection Présentation des activités de l’Institut Pasteur

  12. Overlap of emission spectra from fluorescent dyes used in the LC System • The degree of crosstalk is strongly influenced by: • Temperature • Coupling chemistry of the fluorescence dyes • properties of the optical system Présentation des activités de l’Institut Pasteur

  13. Color compensation activation Without compensation With compensation Présentation des activités de l’Institut Pasteur

  14. Experimental protocol (1) • Chromosomal DNA Extraction Using QIA amp DNA Minikit with : • Isolated culture • Urethral or vaginal samples (stocked in transport medium) • Urines (resuspended in water after centrifugation) • Components of PCR with LightCycler FastStart DNA Master Hybridization Probes Kit (Roche ) Présentation des activités de l’Institut Pasteur

  15. Experimental protocol (2) Présentation des activités de l’Institut Pasteur

  16. Partial sequence alignment L P D Présentation des activités de l’Institut Pasteur

  17. Characteristics of N. gonorrhoeae strains used in the study 120 NG strains Présentation des activités de l’Institut Pasteur

  18. Gene expression at mRNA level Quantitative expression of mRNA encoding pro-inflammatory and immunomodulatory cytokines in hamsters infected with a virulent variant of Leptospira Présentation des activités de l’Institut Pasteur

  19. Cytokines and immunity • Regulatory proteins • Inflammatory responses (TNFa vs IL-10) • Immune and pathological pathways • Why quantifying cytokine expression? • Specific immune mechanisms • Mechanisms of anti-Leptospira immunity • Th1-Th2 profile • Pathogenesis of infection Présentation des activités de l’Institut Pasteur

  20. Syrian hamster (Mesocricetus auratus) • Experimental model • Highly susceptible to Leptospira • Nucleotide sequence of cytokine genes (Melby et al., 1998) Quantification of specific cDNA targets • Absolute quantification • Relative quantification Présentation des activités de l’Institut Pasteur

  21. Definition of used terms Definitions of Frequently Used Terms Term Definition Sample Material of interest (tissue, cells, blood etc.) Target Nucleic acid of interest (specific RNA or DNA sequence) Reference Nucleic acid that is found at a constant copy number in all samples (= endogenous control) The reference is used for normalization of sample-to-sample differences (nucleic acid quality and quantity). Housekeepinggene For mRNA quantification in gene expression studies. A gene that is expressed constitutively on an identical level in all samples to be analyzed. Calibrator A sample that is used for normalization of the final results. The calibrator is positive for target and reference, and must have a constant expression ratio of target to reference. Présentation des activités de l’Institut Pasteur

  22. Absolute quantification Présentation des activités de l’Institut Pasteur

  23. Relative quantification Présentation des activités de l’Institut Pasteur

  24. Normalization Concentration of target (sample) Concentration of reference Calibrator Normalized Ratio = Concentration of target (calibrator) Concentration of reference Présentation des activités de l’Institut Pasteur

  25. Standard Curve Generated with serial dilutions of a standard Présentation des activités de l’Institut Pasteur

  26. From blood to curves • 1 to 2 ml of blood • PBMC purification and in vitro culture • Stimulation of PBMC (LPS, PMA-ionomycin) • Total RNA extraction (High Pure RNA isolation kit, ROCHE) • cDNA preparation (Transcriptor First Strand cDNA synthesis kit, ROCHE) • LightCycler protocol (FastStart SYBR Green) • Specificity : melting curves, gel electrophoresis • cDNA dilutions (2 to 4x107 copies/µl) • Standard curves Présentation des activités de l’Institut Pasteur

  27. Standard curves E= 1,891 E= 1,903 E= 1,899 Ainsi que IL-1, IL-2, IL-4, IL-6, IL-10, IL-12p40 Présentation des activités de l’Institut Pasteur

  28. Set of target and reference genes Set of target and reference genes Cytokine Size Hyb. Temp. Tm IL-2 349 bp 61°C 85.2°C IL-4 342 bp 61°C 87.8°C IL-10 308 bp 61°C 87.8°C IL12p40 308 bp 61°C 89.9°C IFNg 226 bp 60°C 84.6°C TGFb 245 bp 60°C 90.0°C TNFa 278 bp 60°C 89.6°C HPRT 242 bp 60/61°C 82.4/82.9°C b actin 357 bp 60/61°C 90.0/90.4°C Présentation des activités de l’Institut Pasteur

  29. Expression profile of TNF-alpha Présentation des activités de l’Institut Pasteur

  30. Expression profile of IFN-gamma Présentation des activités de l’Institut Pasteur

  31. Expression profile of IL-12p40 Présentation des activités de l’Institut Pasteur

  32. Expression profiles of g IFN and IL-12 + ? Présentation des activités de l’Institut Pasteur

  33. From lymphocyte T to Th1 Présentation des activités de l’Institut Pasteur

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