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BASIC MOLECULAR TECHNIQUES IN INFECTIOUS DISEASES

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Title: BASIC MOLECULAR TECHNIQUES IN INFECTIOUS DISEASES


1
BASIC MOLECULAR TECHNIQUES IN
INFECTIOUS DISEASES
2
Clinical laboratory
  • 1)bacteriology mycology
  • 2)parasitology protozoalogy
  • 3)virology
  • 4)hematology
  • 5)biochemistryhormonmetabolism
  • 6)immunologyserology
  • 7)cytologyhisto-pathology genetics

3
Types of laboratory methods(for infectious
diseases)
  • Direct methods
  • look for/detect the agent
  • Indirect methods
  • detect host response to the agent

4
Ag Ab Reactions
  • PRIMARY
  • IF? RIA ? ELISA ,CLIA
  • SECONDARY
  • Percipitation
  • Agglutination
  • Fulccolation

5
Direct methods(Bacteriologymycology
ParasitologyVirology)
  1. Macroscopic evaluation
  2. Staining
  3. Direct microscopy
  4. Electron microscopy
  5. Rapid tests
  6. Molecular methods
  7. Propagate the agent (culturesensitivity)

No propagation required
6
MOLECULAR TECHNIQUES ADVANTAGES
  • High speed
  • high analytical sensitivity
  • high clinical sensitivity
  • conceptually simple
  • highly specific
  • Amenable to full automation

7
BASIC CATEGORIES OF ANALYSIS USED TO CHARACTERIZE
DNARNA
  • 1)electrophoretic seperations(total,RE,PFGE)
  • 2)hybridization assays
  • 3)amplification techniques (NAAT)
  • 4)restriction fragment length polymorphism(RFLP)
  • 5)sequencing

8
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,..)

9
ELECTROPHORETIC SEPERATION OF NUCLEIC ACIDS
10
RFLP CONCEPT
11
HYBRIDIZATION ASSAY FORMATS
  • 1)liquid or solution phase hybridization
  • 2)solid support hybridization
  • a)DOT/blot(inverse DOT/blot)hybridization
  • b)southernnorthern blot hybridization
  • c)in situ hybridization(tissue,cells,chromosome
    s )
  • d)NA chip technology

12
HYBRIDIZATION CONCEPT
13
SOUTHERNNORTHERN BLOT HYBRIDIZATION
14
FLOURESCENT IN SITU HYBRIDIZATION (FISH)
  • Whole cells or tissue section affixed to glass
    slides.
  • Clinical applications in formalin-fixed paraffin
    embedded tissues.

tissue
15
NA CHIP TECHNOLOGY
16
MICRO ARRAY TECHNOLOGY
17
Application of microarray for pathogen detection
18
DNA SEQUENCING
19
????? ??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

20
principles
21
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22
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

23
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)
24
Multiplex PCR
25
In the field of infectious diseases
  • the technique has been shown to be a valuable
    method for identification of
  • viruses
  • bacteria
  • fungi
  • parasites
  • All

26
REAL TIME PCR
27
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28
APPLICATIONS OF MOLECULAR TECHNIQUES IN MEDICAL
MICROBIOLOGY
29
Microbiology Laboratory
  • Clinical Microbiology comprises essentially 5
    sections.
  • Aerobic and anerobic bacteriology
  • Mycology
  • Mycobacteriology (also called Acid-fast
    Bacteriology, AFB)
  • Parasitology
  • Virology

30
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

31
MOLECULAR METHODS IN CLINICAL BACTERIOLOGY LAB.
  • PCR other amplification techniques
  • nucleic acid hybridization techniques
  • use of RE,s
  • DNA sequencing analysis
  • gene chip technology

32
MOLECULAR METHODS FOR IDENTIFICATION OF
BACTERIA(1)
33
MOLECULAR METHODS FOR IDENTIFICATION OF
BACTERIA(2)
34
MOLECULAR METHODS FOR IDENTIFICATION OF
BACTERIA(3)
35
PCR of M.tuberculosis
36
Molecular detection of mycoplasma
37
PCR DETECTION OF BRUCELLA
38
PCR-based detection of H.pylori
(cag A gene)
39
PCR-based detection of T.pallidum
40
PCR-based detection of Mycobacterium lepre in
skin biopsy
41
PCR-based detection of Yersinia entrocolitica
(chromosomal ail gene)
42
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

43
APPLICATION OF MOLECULAR METHODS IN VIROLOGY
  • Hepatitis viruses(HBV,HCV,HDV)PCRRT-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 PCRRT-PCR
  • rubella RT-PCR

44
QUANTITATIVE AMPLIFICATION RESULTS MAY USEFUL FOR
  • Viral load
  • prognosis
  • monitoring response to therapy

45
HCV RNA ( RT-PCR)
46
QUANTITATIVE AMPLIFICATION FOR
HIV DETECTION
  • branched DNA assay

47
Laboratory Diagnosis of Influenza
Comparison of Test Methods for Influenza Comparison of Test Methods for Influenza Comparison of Test Methods for Influenza
Test Method Time to Results Comments
Serology gt2 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
Antigen Detection (IF) 2-4 hrs Requires reading expertise IF microscope
Antigen Detection (Rapid EIA-like) 15-30 min Widely available, requires little expertise
48
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 2-8C lt 72 hours or freeze at lt -70C.
  • Transport with cool-pack

49
Possible contamination due to the throat- wash
sampling method
50
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51
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52
MOLECULAR DETECTION OF PARASITES PROTOZOA

53
Nested PCR for Leishmania diagnosis
54
PCR-BASED DETECTION OF TOXOPLASMA GONDII
55
MOLECULAR DETECTIONOF FUNGI
  • T.verrocosum HSP 70 PCR
  • Candida sp. PCR
  • Cryptococcus neoformansnested PCR(CSF)
  • Histoplasma capsulatum probe
  • Coccidioides immitis probe
  • Blastomyces dermatidis probe
  • Acanthamoeba PCR

56
Detection of Cryptococcus neoformans by nested PCR
57
MOLECULAR METHODS FOR
CO-IDENTIFICATION OF MULTIPLE AGENTS
58
Clinical manifestation(s) and/or specimen Pathogens targeted Infectious agent
Genital ulcer disease HSV, H. ducreyi, and T. pallidum Combination (All agents)
Genital swabs HPVs, HSV, and C. trachomatis
Keratoconjunctivitis Adenovirus, HSV, and C. trachomatis
Acute respiratory tract infection EV, influenza viruses A and B, RSV, PIV types 1 and 3, adenovirus, M. pneumoniae, and C. pneumoniae
59
Application of multiplex PCR for diagnosis of
viral infections(viruses in CNS)
Viruses and/or other agent(s) targeted Specimen(s) Clinical manifestation(s)
HSV-1, HSV-2, and CMV HSV and VZV EBV and HHV-6 HSV-1, HSV-2, VZV, CMV, HHV-6, and EBV CSF Meningitis, encephalitis, and/or meningoencephalitis
HSV-1, HSV-2, VZV, CMV, HHV-6, EBV, and Ent.V six herpesviruses that may infect the CNS
CMV, EBV, HHV-6, HHV-7, and HHV-8

60
16 S rDNA
61
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
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