Pr

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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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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

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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

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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

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

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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
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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
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Tm and mutation detection
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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

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Color compensation activation
Without compensation
With compensation
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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 )

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Experimental protocol (2)
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Partial sequence alignment
L
P
D
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Characteristics of N. gonorrhoeae strains used in
the study
120 NG strains
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Gene expression at mRNA level
Quantitative expression of mRNA encoding
pro-inflammatory and immunomodulatory cytokines
in hamsters infected with a virulent variant of
Leptospira
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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

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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

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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
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Absolute quantification
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Relative quantification
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Normalization
Concentration of target
(sample)
Concentration of reference
Calibrator Normalized Ratio

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

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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
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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
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Expression profile of TNF-alpha
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Expression profile of IFN-gamma
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Expression profile of IL-12p40
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Expression profiles of g IFN and IL-12
?
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From lymphocyte T to Th1