Download MOLECULAR TECHNIQUES IN LABORATORY DIAGNOSIS

BASIC MOLECULAR TECHNIQUES
IN
INFECTIOUS DISEASES
Dr.Sarookhani
Clinical laboratory
•
•
•
•
•
•
•
1)bacteriology & mycology
2)parasitology & protozoalogy
3)virology
4)hematology
5)biochemistry&hormon&metabolism
6)immunology&serology
7)cytology&histo-pathology & genetics
Dr.Sarookhani
Types of laboratory methods
(for infectious diseases)
• Direct methods
– look for/detect the agent
• Indirect methods
– detect host response to the agent
Dr.Sarookhani
Ag Ab Reactions
• PRIMARY
• IF٬ RIA ٬ ELISA ,CLIA
• SECONDARY
• Percipitation
• Agglutination
• Fulccolation
Dr.Sarookhani
Direct methods
(Bacteriology&mycology& Parasitology&Virology)
1.
2.
3.
4.
5.
6.
7.
Macroscopic evaluation
Staining
Direct microscopy
No propagation
required
Electron microscopy
Rapid tests
Molecular methods
Propagate the agent (culture&sensitivity)
Dr.Sarookhani
MOLECULAR TECHNIQUES
ADVANTAGES
•
•
•
•
•
•
High speed
high analytical sensitivity
high clinical sensitivity
conceptually simple
highly specific
Amenable to full automation
Dr.Sarookhani
BASIC CATEGORIES OF
ANALYSIS USED TO
CHARACTERIZE DNA&RNA
•
•
•
•
•
1)electrophoretic seperations(total,RE,PFGE)
2)hybridization assays
3)amplification techniques (NAAT)
4)restriction fragment length polymorphism(RFLP)
5)sequencing
Dr.Sarookhani
LABORATORY SPECIMENS FOR
MOLECULAR TECHNIQUES
• 1)whole blood& PBMC
• 2)serum
• 3)body fluids (urine, semen,
CSF,ameniotic fluid,...)
• 4)biopsies
• 5) placenta & CVS
• 6)blastomer cells of embryo
• 7)others(hair,stool,smears,..)
Dr.Sarookhani
ELECTROPHORETIC
SEPERATION OF
NUCLEIC ACIDS
Dr.Sarookhani
RFLP CONCEPT
Dr.Sarookhani
HYBRIDIZATION ASSAY
FORMATS
1)liquid or solution phase hybridization
2)solid support hybridization
a)DOT/blot(&inverse DOT/blot)hybridization
b)southern&northern blot hybridization
c)in situ hybridization(tissue,cells,chromosomes )
d)NA chip technology
Dr.Sarookhani
HYBRIDIZATION CONCEPT
Dr.Sarookhani
SOUTHERN&NORTHERN BLOT
HYBRIDIZATION
Dr.Sarookhani
FLOURESCENT IN SITU
HYBRIDIZATION (FISH)
– Whole cells or tissue section affixed to glass slides.
– Clinical applications in formalin-fixed paraffin embedded tissues.
tissue
Dr.Sarookhani
NA CHIP TECHNOLOGY
Dr.Sarookhani
MICRO ARRAY
TECHNOLOGY
Dr.Sarookhani
Application of microarray for
pathogen detection
Dr.Sarookhani
DNA SEQUENCING
Dr.Sarookhani
Nucleic Acid Amplification
Technologies (NAAT)
• 1)TARGET AMPLIFICATION METHODS
a)PCR & modifications
b)NASBA
c)TMA
d)SDA
• 2)PRIMER(PROB) AMPLIFICATION METHODS
a)LCR
b)Q-beta replicase
c)cleavase / invader technology
• 3)SIGNAL AMPLIFICATION METHODS
a)b DNA
&
b)HCA
Dr.Sarookhani
principles
Dr.Sarookhani
Dr.Sarookhani
PCR-based modification techniques
•
•
•
•
•
•
•
•
•
•
•
RT-PCR
nested PCR
hot start PCR
PCR-LiPA
PCR-SSP
PCR-ARMS
PCR-RFLP
multiplex PCR
PCR-SSCP
RACE-PCR
Real time PCR
Dr.Sarookhani
Schematic of Multiplex PCR
In multiplex PCR more than one target sequence can be amplified by including more than one
pair of primers in the reaction. ( Amplifying various genes simultaneously)
Locus A
Locus
C
Locus
B
A
B
C
small
large
Dr.Sarookhani
Multiplex PCR
Dr.Sarookhani
In the field of
infectious diseases
the technique has been shown to be a valuable
method for identification of:
• viruses
• bacteria
• fungi
• parasites
• All
Dr.Sarookhani
REAL TIME PCR
Dr.Sarookhani
Dr.Sarookhani
APPLICATIONS OF MOLECULAR
TECHNIQUES IN MEDICAL
MICROBIOLOGY
Dr.Sarookhani
Microbiology Laboratory
•
Clinical Microbiology comprises essentially 5 sections.
• Aerobic and anerobic bacteriology
• Mycology
• Mycobacteriology (also called Acid-fast
Bacteriology, AFB)
• Parasitology
• Virology
Dr.Sarookhani
IMPACT OF MOLECULAR
METHODS ON CLINICAL
BACTERIOLOGY
• 1)DIAGNOSIS & PATHOGEN IDENTIFICATION
a)for slow growing or difficult-to- culture organisms
b)further examination & identification of agar-grown
pure cultures
c)simultaneous isolation of pathogens from
specimens
• 2)THERAPY
• 3)EPIDEMIOLOGY & CONTROL MEASURES
Dr.Sarookhani
MOLECULAR METHODS IN
CLINICAL BACTERIOLOGY LAB.
•
•
•
•
•
PCR & other amplification techniques
nucleic acid hybridization techniques
use of RE,s
DNA sequencing analysis
gene chip technology
Dr.Sarookhani
MOLECULAR METHODS FOR
IDENTIFICATION OF BACTERIA(1)
Streptococci group
Group A streptococci
Pneumococci
N.gonorrhea
N.gonorrhea
N.gonorrhea
N.meningitidis
Entrococci
Staphylococci
Staphylococci
16S rRNA
Emm gene
Lyt A gene
rRna
CPPB gene
Pillin gene
rrn gene
rRNA
HSP60
Nuc&femA
RFLP
Hybridizat&PCR
DNAprobe&PCR
DNA probe
PCR
LCR
PCR
Probe
Colony DOT blot
PCR Dr.Sarookhani
MOLECULAR METHODS FOR
IDENTIFICATION OF BACTERIA(2)
H.influenza
PBP& rRNA
Probe
H.influenza
BexA&ompP6 PCR
Legionella
rRNA
Probe
Legionella
mip
PCR
H.ducreyi
16 S rRNA
Probe
H.ducreyi
groEL
PCR
Mycoplasma&Ureaplasma
MP & MCC
Probe
Mycoplasma&Ureaplasma
Tuf&urease
PCR
Dr.Sarookhani
MOLECULAR METHODS FOR
IDENTIFICATION OF BACTERIA(3)
H.pylori
Ure A,C&rRNA Probe&PCR
Y.entrocolytica
ail
Cholera toxin
Elt&ctx&hlyA PCR
C.diphteria
toxA
Probe
M.tuberculosis
IS
probes
M.tuberculosis
IS6110
PCR&RFLP
&LCR
Dr.Sarookhani
PCR
PCR of M.tuberculosis
Dr.Sarookhani
Molecular detection of
mycoplasma
Dr.Sarookhani
PCR DETECTION OF BRUCELLA
Dr.Sarookhani
PCR-based detection of H.pylori
(cag A gene)
Dr.Sarookhani
PCR-based detection of T.pallidum
Dr.Sarookhani
PCR-based detection of
Mycobacterium lepre in
skin biopsy
Dr.Sarookhani
PCR-based detection of Yersinia
entrocolitica (chromosomal ail gene)
Dr.Sarookhani
APPLICATIONS OF
MOLECULAR EPIDEMIOLOGY
• Detection of identity of strains
• detection of genotypes
• detection of emergence & spread of strains
of an organism with unusual resistance
patterns or pathogenicity
• determining the efficiency of infection
control procedures
• identification of source in outbreaks
Dr.Sarookhani
APPLICATION OF MOLECULAR
METHODS IN VIROLOGY
•
•
•
•
•
•
•
•
Hepatitis viruses(HBV,HCV,HDV):PCR&RT-PCR
herpesviridae(CMV,HSV,EBV,VZV,...):PCR
HTLV1 & HIV1,2, : nested RT-PCR
ENTOVIRUSES :RT-PCR
PARVOVIRUS B19 : HB & PCR
HPV : FISH
mumps,adenovridae,LCM,measles : PCR&RT-PCR
rubella : RT-PCR
Dr.Sarookhani
QUANTITATIVE
AMPLIFICATION RESULTS
MAY USEFUL FOR:
• Viral load
• prognosis
• monitoring response to therapy
Dr.Sarookhani
HCV RNA ( RT-PCR)
Dr.Sarookhani
QUANTITATIVE
AMPLIFICATION
FOR HIV DETECTION
•
• branched DNA assay
Dr.Sarookhani
Laboratory Diagnosis of Influenza
Comparison of Test Methods for Influenza
Test Method
Time to
Results
Comments
Serology
>2 wks
Retrospective, requires paired sera
Culture
1-10 days
Still gold standard(?), requires
expertise, provides virus for studies
Molecular
(RT-PCR)
2-4 hrs
Becoming gold standard(?), requires
expertise & expensive equipment
2-4 hrs
Requires reading expertise & IF
microscope
15-30 min
Widely available, requires little
expertise
Antigen Detection
(IF)
Antigen
Detection
(Rapid EIA-like)
Dr.Sarookhani
Specimen Types
• Upper respiratory tract
– Nasal or naso-pharyngeal (NP)
swabs
– Throat swabs
– NP aspirates or washes
• Lower respiratory tract
– Tracheal aspirates
– Bronchoalveolar lavages
• Store at 2-8°C < 72 hours or
freeze at < -70°C.
– Transport with cool-pack
Dr.Sarookhani
Possible contamination due to the throat- wash sampling
method
Dr.Sarookhani
Dr.Sarookhani
Dr.Sarookhani
MOLECULAR DETECTION
OF PARASITES &PROTOZOA
Hydatidosis
(protoscolex)
r-DNA
ITS 1
PCR-RFLP
•
Plasmodium sp.
A.culcifacies
Mt-DNA
toxoplasmosis
B1
E.histolytica &
(dispar var.)
r RNA
leishmaniosis
Dr.Sarookhani
Nested
PCR-RFLP
PCR-RFLP
DOT/BLOT&
PCR
Hybridization
& PCR
PCR
Nested PCR for Leishmania
diagnosis
Dr.Sarookhani
PCR-BASED DETECTION OF
TOXOPLASMA GONDII
Dr.Sarookhani
MOLECULAR DETECTION
OF FUNGI
•
•
•
•
•
•
•
T.verrocosum :HSP 70 :PCR
Candida sp. :PCR
Cryptococcus neoformans:nested PCR(CSF)
Histoplasma capsulatum: probe
Coccidioides immitis :probe
Blastomyces dermatidis :probe
Acanthamoeba :PCRDr.Sarookhani
Detection of Cryptococcus
neoformans by nested PCR
Dr.Sarookhani
MOLECULAR METHODS FOR
CO-IDENTIFICATION OF
MULTIPLE AGENTS
Dr.Sarookhani
Infectious agent
Combination
(All agents)
Pathogens targeted
Clinical manifestation(s) and/or specimen
HSV,
Genital ulcer disease
HPVs,
HSV, and
Genital swabs
H. ducreyi, and
T. pallidum
C. trachomatis
Adenovirus,
HSV, and
Keratoconjunctivitis
C. trachomatis
EV, influenza viruses A and
Acute respiratory tract
B, RSV, PIV types 1 and 3,
infection
adenovirus, M. pneumoniae,
and C. pneumoniae
Dr.Sarookhani
Application of multiplex PCR for diagnosis of viral infections
(viruses in CNS)
Clinical manifestation(s)
•Meningitis, encephalitis,
and/or meningoencephalitis
Specimen(s)
CSF
six herpesviruses
Viruses and/or other
agent(s) targeted
HSV-1, HSV-2, and CMV
HSV and VZV; EBV and HHV-6
HSV-1, HSV-2, VZV, CMV, HHV-6,
and EBV
HSV-1, HSV-2, VZV, CMV,
HHV-6, EBV, and Ent.V
that may infect the CNS
CMV, EBV, HHV-6, HHV-7, and
HHV-8
Dr.Sarookhani
16 S rDNA
Dr.Sarookhani
Advantages of Molecular techniques
in Infectious Diseases
•
•
•
•
•
•
•
increased sensitivity and specificity of
identification
faster report turnaround time
Confirmation of culture
Identification of organisms that are non-viable or
cannot be cultured
Identification of fastidious, slow growing
organisms
Identification of organisms that are dangerous to
culture
Identification of organisms in small numbers or
in small volume specimens
Dr.Sarookhani