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Usually symptomatic in males, often asymptomatic in women ... Specificity : melting curves, gel electrophoresis. cDNA dilutions (2 to 4x107 copies/ l) ... – PowerPoint PPT presentation

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Title: Prsentation PowerPoint


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

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

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

5
b-lactams action mode
Antibiotics
Penicillin binding proteins
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
7
Mechanisms of penicillin resistance in
N.gonorrhoeae (2)
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

9
Oligonucleotide sequences
Design with Probe design software 2.0 (Roche
Diagnostics)
a GenBank accession number b LC-R705,
LightCycler red 705 c LC-R640, LightCycler
red 640
10
FRET based-system for the detection of ponA
mutation (1)
T
C
or
881 bp
3
5
ponA-F
ponA-P2
ponA-P1
LC705
FL
3
5
GGCGTTGGGCGGTGGT
AAGAGCCGTTGCTGCA(PO4)
A
814
1232
1265
D
h?
ponA-R
FRET
5
1679
3
5
Reverse Primer
Anchor Probe
Sensor Probe or Detection Probe
Forward Primer
11
Tm and mutation detection
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

13
Color compensation activation
Without compensation
With compensation
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 )

15
Experimental protocol (2)
16
Partial sequence alignment
L
P
D
17
Characteristics of N. gonorrhoeae strains used in
the study
120 NG strains
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
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

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

21
Definition of used terms
Definitions of Frequently Used Terms
Term
Definition
Material of interest (tissue, cells, blood etc.)
Sample
Nucleic acid of interest (specific RNA or DNA
sequence)
Target
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).
Reference
For mRNA quantification in gene expression
studies.
Housekeeping gene
A gene that is expressed constitutively on an
identical level in all samples to be analyzed.
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.
Calibrator
22
Absolute quantification
23
Relative quantification
24
Normalization
Concentration of target
(sample)
Concentration of reference
Calibrator Normalized Ratio

Concentration of target
(calibrator)
Concentration of reference
25
Standard Curve
Generated with serial dilutions of a standard
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

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
28
Set of target and reference genes
Set of target and reference genes
Cytokine Size Hyb. Temp. Tm IL-2 349
bp 61C 85.2C IL-4 342 bp 61C 87.8C IL-10 3
08 bp 61C 87.8C IL12p40 308 bp 61C 89.9C IFN
g 226 bp 60C 84.6C TGFb 245
bp 60C 90.0C TNFa 278 bp 60C 89.6C HPRT 24
2 bp 60/61C 82.4/82.9C b actin 357
bp 60/61C 90.0/90.4C
29
Expression profile of TNF-alpha
30
Expression profile of IFN-gamma
31
Expression profile of IL-12p40
32
Expression profiles of g IFN and IL-12
?
33
From lymphocyte T to Th1
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