Post on 11-Jan-2016
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. Macroscopic evaluation
2. Staining
3. Direct microscopy
4. Electron microscopy
5. Rapid tests
6. Molecular methods
7. Propagate the agent (culture&sensitivity)
No propagation required
Dr.Sarookhani
MOLECULAR TECHNIQUES ADVANTAGES
• High speed
• high analytical sensitivity
• high clinical sensitivity
• conceptually simple
• highly specific
• Amenable to full automationDr.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.
tissueDr.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)(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
C
B
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 MOLECULAR TECHNIQUESTECHNIQUES 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 16S rRNA RFLPGroup A streptococci Emm gene Hybridizat&PCRPneumococci Lyt A gene DNAprobe&PCRN.gonorrhea rRna DNA probeN.gonorrhea CPPB gene PCRN.gonorrhea Pillin gene LCRN.meningitidis rrn gene PCREntrococci rRNA ProbeStaphylococci HSP60 Colony DOT blotStaphylococci Nuc&femA 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 PCRMycoplasma&Ureaplasma MP & MCC ProbeMycoplasma&Ureaplasma Tuf&urease PCR
Dr.Sarookhani
MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(3)
H.pylori Ure A,C&rRNA Probe&PCR
Y.entrocolytica ail PCR
Cholera toxin Elt&ctx&hlyA PCR
C.diphteria toxA Probe
M.tuberculosis IS probes
M.tuberculosis IS6110 PCR&RFLP&LCR
Dr.Sarookhani
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 identityidentity of strains
• detection of genotypesgenotypes
• detection of emergence & spread of strains of an organism with unusualunusual resistance patterns or pathogenicity
• determining the efficiency efficiency of infection control procedures
• identification of sourcesource in outbreaksDr.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-PCRDr.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 forComparison of Test Methods for InfluenzaInfluenza
Test MethodTest MethodTime to Time to ResultsResultsCommentsComments
SerologySerology>>22 wkswksRetrospective, requires paired seraRetrospective, requires paired sera
CultureCulture1-101-10 daysdaysStill gold standard(?), requires Still gold standard(?), requires expertise, provides virus for studiesexpertise, provides virus for studies
MolecularMolecular((RT-PCRRT-PCR))2-42-4 hrshrsBecoming gold standard(?), requires Becoming gold standard(?), requires
expertise & expensive equipmentexpertise & expensive equipment
Antigen Detection Antigen Detection )IF()IF(2-42-4 hrshrsRequires reading expertise & IF Requires reading expertise & IF
microscopemicroscope
Antigen DetectionAntigen Detection ((Rapid EIA-likeRapid EIA-like))15-3015-30 minminWidely available, requires little Widely available, requires little
expertiseexpertiseDr.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-DNAITS 1
PCR-RFLP
Plasmodium sp. NestedPCR-RFLP
A.culcifacies Mt-DNA PCR-RFLP
toxoplasmosis B 1 DOT/BLOT&PCR
E.histolytica &(dispar var.)
r RNA Hybridization& PCR
leishmaniosis PCR
Dr.Sarookhani
Nested PCR for Leishmania diagnosis
Dr.Sarookhani
PCR-BASED DETECTION OF TOXOPLASMA GONDII
Dr.Sarookhani
MOLECULAR DETECTIONOF 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 agentInfectious agent Pathogens targetedPathogens targeted Clinical manifestation(s) and/or Clinical manifestation(s) and/or
specimenspecimen
CombinationCombination
((All agentsAll agents))
HSV, HSV, H. ducreyiH. ducreyi, and , and T. pallidumT. pallidum
Genital ulcer disease Genital ulcer disease
HPVs, HPVs, HSV, and HSV, and C. trachomatisC. trachomatis
Genital swabsGenital swabs
Adenovirus, Adenovirus, HSV, and HSV, and C. trachomatisC. trachomatis
Keratoconjunctivitis Keratoconjunctivitis
EV, influenza viruses A EV, influenza viruses A and B, RSV, PIV types 1 and B, RSV, PIV types 1 and 3, adenovirus, and 3, adenovirus, M. M. pneumoniaepneumoniae, and , and C. C. pneumoniaepneumoniae
Acute respiratory Acute respiratory tract infectiontract infection
Dr.Sarookhani
Application of multiplex PCR for diagnosis of viral infections
(viruses in CNS)
•
Clinical manifestation(s)Clinical manifestation(s) Specimen(s)Specimen(s) Viruses and/or other Viruses and/or other
agent(s) targetedagent(s) targeted
Meningitis, encephalitis, Meningitis, encephalitis, and/or and/or
meningoencephalitismeningoencephalitis CSFCSFHSV-1, HSV-2, and CMV HSV-1, HSV-2, and CMV
HSV and VZV; EBV and HHV-6 HSV and VZV; EBV and HHV-6 HSV-1, HSV-2, VZV, CMV, HSV-1, HSV-2, VZV, CMV,
HHV-6, and EBVHHV-6, and EBV
six herpesvirusessix herpesviruses
that maythat may infect the CNS infect the CNS
HSV-1, HSV-2, VZV, CMV, HSV-1, HSV-2, VZV, CMV, HHV-6, EBV, and Ent.V HHV-6, EBV, and Ent.V
CMV, EBV, HHV-6, HHV-7, CMV, EBV, HHV-6, HHV-7,
and HHV-8and 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 specimensDr.Sarookhani