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Communicable Diseases I

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Title: Communicable Diseases I


1
Communicable Diseases I II
  • Dr U L Fairbrother

2
Communicable Disease Agents Are.?
  • Name four

3
Communicable Disease Agents Are
  • Bacteria
  • Fungi
  • Protozoa
  • Viruses

4
Communicable Disease Agents Cause..
  • E.g.
  • MRSA (Methicillin resistant Staphylococcus
    aureus), TB (Mycobacterium tuberculosis) and
    leprosy (Mycobacterium leprae)
  • Candidiasis (Candida albicans or Pneumonia
    (Pneumocystis carinii)
  • Malaria (Plasmodium falciparum, Plasmodium vivax,
    Plasmodium ovale, Plasmodium malaria )
  • HIV/AIDS (Human Immunodeficiency virus/Aquired
    Immunodeficiency Syndrome)

5
Diagnostic Methods for Bacterial Infections
  • Older (non-molecular techniques) usually used
  • Bacteria cultured phenotypic characterisation
  • Conditions in which they grow
  • Morphology
  • Gram staining from cultured colonies
  • Biochemical tests
  • Some examples to follow

6
Gram Stain of BacteriaGram Positive
  • Bacteria that up take the original purple dye
    only have a cell wall
  • Eg Staphylococcus epidermis
  • Causes boils

7
Gram Negative
  • Bacteria that lose purple dye and can therefore
    take up the second red dye have both a cell wall
    and a cell membrane
  • Eg Escherichia coli
  • normally benign, ubiquitous, gut-dwelling

8
Gram Staining Video
  • http//www-micro.msb.le.ac.uk/Video/gram.mov

9
Biochemical Tests
  • relatively few commercially available molecular
    methods for the identification of clinically
    significant gram-negative bacilli in the clinical
    laboratory exist today
  • the need for identification procedures that use
    more conventional processes remains.
  • Some of these phenotypic identification
    procedures are based on colorimetric or pH-based
    changes and usually require 18 to 24 h to
    identify organisms.
  • Some are based on changes in preformed enzymes,
    shortening to 2 to 4 h the time necessary to make
    an identification.

10
CITRATE TEST
  • Ability of bacterium to utilise citrate as source
    of carbon.
  • Breaks citrate into organic acids and carbon
    dioxide.
  • CO2 combines with sodium, forming sodium
    carbonate.
  • A pH indicator detects the presence of this
    compound by turning blue (a positive test).

11
COAGULASE TEST
  • Differientiates between pathogenic and
    non-pathogenic strains of Staphylococcus.
  • Coagulase-defense mechanism, clots area of plasma
    around them
  • Resists phagocytosis by the host's immune system.

12
OPTOCHIN TEST
  • Identify strains of Streptococcus pneumoniae.
  • ethyl hydrocupreine disks placed on inoculated
    blood agar plates.
  • a zone of inhibition will develop around the disk
    where the bacteria have been lysed.

13
Molecular methods
  • New technologies enable microbiology results to
    be available in minutes or hours rather than days
  • M.tuberculosis, Bacillus anthracis, Salmonella
    spp. and Shigella dysenteriae
  • examples of bacteria which require high level
    containment facilities to grow them - dangerous!
  • Better if they could be inactivated then perform
    PCR for diagnosis / analysis

14
Rapid Accurate Diagnosis is Advantageous
  • Earlier institution of therapy reduces infection
    related morbidity and mortality
  • May prevent rapid dissemination of an epidemic
  • Narrower spectrum of antimicrobial agents used
    earlier reducing cost and adverse effects
  • Invasive diagnostic procedures

15
Importance of Immunoassays to Health
  • The immunoassay is the workhorse of analytical
    biochemistry
  • unique binding abilities of antibodies to be
    widely used in selective and sensitive
    measurement of small and large molecular analytes
    in complex samples.
  • used in two general classes of diagnostic
    applications 1) the diagnosis of a disease state
    or identification of the organism responsible for
    a disease state, and 2) the management of
    treatment for a disease, either through
    monitoring of the disease state or of the drugs
    used for therapy.

16
Immunoassays Usefulness
  • Technical simplicity, rapidity, specificity, and
    cost effectiveness
  • dipstick-type formats possible (New Rapid
    Diagnostic Tests for Neisseria meningitidis
    Serogroups A, W135, C, and, Chanteau et al., PLoS
    Med. 2006 September 3(9) e337. )
  • Mainly restricted to centralized laboratories
    because of the need for long assay times, complex
    and expensive equipment, and highly trained
    technicians.
  • but can have poor sensitivity and low negative
    predictive value

17
(No Transcript)
18
How Immunoassays Work
  • Exploit highly selective binding between antibody
    and antigen.
  • Some in homogeneous phases (in solution or gels),
    most heterogeneous assays - adhesion of
    antibodies and/or antigens to a solid surface.
    E.g. walls of microtitre plates
  • Some competitive labeled analytes compete for
    the binding sites of antibodies.
  • decrease in bound, labeled antigen is measured.
  • more sensitive assays "sandwich" the analyte
    between an immobilized primary and mobile but
    labeled secondary antibody.

19
Enzyme-linked immunosorbent assay
  • It employs an enzyme label for detection of
    antibodyantigen complexes formed on a solid
    phase.
  • Detection of antibodyantigen complexes based on
    the enzyme catalytic activity of an appropriate
    colorless substrate to give a colored product,
    whose intensity is measured by the optical
    density.

20
ELISA
Autoimmune and infectious diseases diagnosed
using ELISA Coeliac diseaseImmune reactions to
foodInfectious reactions to foodInfectious
diseases such as syphilis and TBLupus
diseasePernicious aneamiaSystemic rheumatic
disease ThrombosisThyroid diseaseRenal disease
  • the ELISA is one of the most widely used
    immunodiagnostic tools, particularly in
    diagnosing infectious disease
  • Gosling 1990

21
ELISA Animation
  • www.immunospot.com/elisa-animation.html

22
Molecular Genetics
  • Viruses, bacteria, fungi, and protozoa can be
    detected and characterised by molecular
    biological methods
  • Some commercial kits use molecular techniques for
    diagnosis, e.g. for TB, Legionella pneumophila.
  • Number of amplicons indicated by a colour change
    (using e.g. HPO and tetramethylbenzidine).
  • Genotyping is also used for epidemiological
    reasons, for tracking an outbreak and locating
    the source of e.g. E.coli 0157H7

23
Molecular Characterisation of Bacteria
  • strain typing and resistance genotyping useful
    for pathogenic microorganisms worldwide.
  • Resistance phenotypes include multidrug-resistant
    pathogens, extended-spectrum -lactamase
    (ESBL)producing Enterobacteriaceae,
    methicillin-resistant Staphylococcus aureus
    (MRSA), vancomycin-resistant enterococci, and
    fluoroquinolone-resistant (FQR) strains of
    gram-negative bacilli and Streptococcus
    pneumoniae.

24
PCR
  • Using universal or specific primers AND
    identification of amplicon by sequencing -rapid
    identification of cultured/uncultured bacteria.
  • Quick diagnosis of fastidious pathogens for which
    culture difficult. e.g. Mycobacterium
    tuberculosis (TB), Mycobacterium leprae
    (leprosy).
  • Pitfalls, such as false positives, interpret the
    results with caution.
  • Bacterial genome sequencing and real-time PCR
    increased speed, simplicity, reproducibility,
    quantitative capability and lower risk of
    contamination.

25
PCR problems
  • Cross over contamination.
  • Cost
  • PCR Inhibitors (e.g. Haemoglobin is a potent
    inhibitor of Taq polymerase)
  • Positive results do not always have biological
    significance, i.e., PCR reacts with inactivated
    pathogens e.g. viruses, as well as infectious
    viruses.

26
PCR advantages
  • Speed
  • Dead or alive
  • Sensitivity
  • For pathogens, molecular techniques are fast,
    sensitive, specific, and quantifiable compared
    with e.g. immunological techniques.
  • avoids the risk, and time taken, to grow up
    viruses in cells in vitro.

27
Interactive LabBacterial ID lab
  • http//www.hhmi.org/biointeractive/disease/vlab.ht
    ml
  • Split into six groups of four and do one step per
    group

28
RT PCR
  • RT-PCR provides a sensitive and rapid detection
    and has facilitated the typing and subtyping of
    viruses.
  • Previously, researchers developed tests to detect
    H5N1 virus by using conventional RT-PCR and
    confirmed the results by Southern blot analysis.
  • Currently use e.g. real-time PCR successfully.

29
Mechanics of RT PCR
  • Reverse transcribed with reverse trancriptase,
    dNTP and random primers
  • PCR primers dNTPs and Taq polymerase
  • First strand is a duplex.

30
RT PCR Animation
  • http//www.bio.davidson.edu/Courses/genomics/RTPCR
    /RT_PCR.html

31
Characterisation for Resistance Genotype
  • by PCR and DNA sequencing.
  • Identifies clonal spread in clusters of
    multiresistant pathogens.
  • E.g MRSA, Enterobacteriaceae, ESBL-producing
    strains of Escherichia coli, Klebsiella
    pneumoniae ,Pseudomonas aeruginosa, Acinetobacter
    species, and Stenotrophomonas maltophilia
  • phenotypic and genotypic characterisation is
    powerful, providing information important for
    global antimicrobial surveillance.

32
PCR Based method for MRSA
  • rapid identification of methicillin-resistant
    Staphylococcus aureus (MRSA) are based on the
    detection of an S. aureus-specific gene target
    and the mecA gene
  • Multiplex PCR Strategy for Rapid Identification
    of Structural Types and Variants of the mec
    Element in Methicillin-Resistant Staphylococcus
    aureus (2002) Oliveira DC, de Lencastre H.
    Antimicrob Agents Chemother. 46 21552161.

33
Validation of the SCCmec multiplex PCR strategy
  • Major SCCmec types at the bottom. (A) SCCmec type
    I (lanes 1 to 4 and 8), variant IA (lanes 5 to 7
    and 9), and SCCmec type II (lanes 10 to 12). (B)
    SCCmec type III (lanes 1, 2, 6, and 7), variant
    IIIA (lanes 3 and 4), and variant IIIB (lane 5).
    (C) SCCmec type IV. M, DNA molecular size marker

34
Fungal infections
  • Traditional methods widely used - rapid diagnosis
    not usually necessary
  • use molecular methodology for detecting
    Pneumocystis carinii in suspected HIV cases.
  • Candida spp. important in immunosuppressed
    patients

35
Detection of seven Candida species using the
Light-Cycler system P. Lewis White, Anjali
Shetty and Rosemary A. Barnes
  • detect, but not differentiate between, seven
    species of Candida (Candida albicans, Candida
    dubliniensis, Candida glabrata, Candida kefyr,
    Candida krusei, Candida parapsilosis and Candida
    tropicalis).
  • Single-round amplification allowed rapid
    turn-around of clinical samples (within one
    working day)
  • more sensitive, exposing 39 possible systemic
    infections not detected by blood culture.
  • Med Microbiol 52 (2003), 229-238

J
36
Protozoa
  •  not widely used, except for
  •  assessing drug-resistance in Plasmodium spp.
    (four of these cause malaria).
  • Cryptosporidium spp and Naegleria spp.
    contamination of water supplies.
  • All molecular tests are expensive, yet many of
    these diseases occur in places where they cannot
    be afforded (nor can the treatments!).
  • Increased automation and development of kits may
    help to decrease costs in the future.

37
Restriction Enzyme Digestion of Protozoan PCR
Product
  • Human type of Cryptosporidium parvum causes
    waterborne life threatening diarrhoea, in AIDS
    patients.
  • PCR-RFLP discriminates between Human and Bovine
    Genotype Isolates,
  • Samples 1 , 2 9,10,11 are human isolate
    genotype, 3,4,5,7 and 8 are bovine.
  • Restriction fragment length polymorphism
  • amplification of target sequence and digestion

38
Evaluation of Reverse Transcription-PCR Assays
for Rapid Diagnosis of Severe Acute Respiratory
Syndrome Associated with a Novel Coronavirus
  • April 2003, 1,500 cases of SARS in Hong Kong.
  • Rapid confirmation of SARS CoV infection vital
  • serological testing used for retrospective
    diagnosis
  • diagnosis of the infection in the early phase of
    the illness was important for patient care.
  • first-generation reverse transcription (RT)-PCR
    assays were used during this outbreak as
    molecular diagnostic methods for SARS CoV

39
Viruses and Virus isolation (VI)
  • conventionally involves 
  • Recovery of virus
  • Identification of the isolate using in vitro cell
    culture by
  • immunofluorescence microscopy
  • ELISA
  • electron microscopy
  • molecular techniques.

40
VI attempted under specific circumstances
  • Laborious, expensive, potentially hazardous, and
    time consuming (1-2 weeks) 
  • When other detection methods fail or when trying
    to isolate virus(es) from previously unrecognized
    diseases.
  • If there is no other detection method of similar
    or greater sensitivity.
  • If the virus is required for other purposes, such
    as differentiation, characterization, production
    of vaccines.

41
Proper handling of specimens is critical for VI.
  • Can be done on most clinical specimens,
    including
  • biopsy and necropsy tissues
  • blood
  • secretions
  • excretions.
  • Urine, faeces, semen difficult to work with
    because they are toxic to cell cultures.

42
Molecular Techniques for Viruses
  • For viral work, molecular techniques are fast,
    sensitive, specific, and quantifiable compared
    with e.g. immunological techniques.
  • It also avoids the risk, and time taken, to grow
    up viruses in cells in vitro.
  • Viral load is the best single prognostic
    indicator in HIV infection is
  • measured by molecular methods such as PCR e.g
    Quantitative Competitive PCR.

43
Molecular Quantitation of PathogensDNA Probe
Hybridisation
  • Labelled DNA (or RNA ) sequence will anneal to a
    complementary sequence. The probe is used to
    detect the presence of complementary sequences.  
  • If the probe binds to the membrane (or tissue),
    this confirms that a sequence complementary to
    the probe is present on the membrane.
  • Less sensitive than PCR.
  • Probes often used in combination with PCR, PCR
    providing enhanced sensitivity, probe providing
    the specificity.

44
Quantitative viral estimation
  •  Quantitative Competitive PCR uses an internal
    control (a template) which is amplified as
    efficiently as the target sequence.
  • A known amount of DNA fragment (competitor "C")
    is added to the sample.
  • This must contain sequences for the same primers
    as target ("T") DNA.
  • After PCR, run products on a gel.

45
Quantitative viral estimation cont..
  • Ratio of the amounts of the two amplified
    products (amplicons) reflects the ratio of the
    amounts of target DNA and competitor.
  • Initial amount of added competitor is known -so
    the amount of target DNA can be estimated
    according to the TC ratio. 
  • T amount of amplified product from target.
  • C amount of amplified product from competitor.
  • When TC 1, we then know amount of target DNA.

46
Schematic of Quantitative Estimation
47
Calculating Copy Number
  • Amplified by the same primers as target
  • Distinguishable from target
  • Quantity is known
  •  We know weight (e.g. by assay)
  •   We know no. of bp, so can calculate RMM
  •  Therefore we know copy number (no. of
    molecules).

48
Worked Example
  • Say 1.0 ng DNA added
  • if no. of base pairs of competitor 1000 then
    RMM 330,000 Daltons
  • so no. of moles 1x10-9 3x10-15 moles
  • 330,000
  • Using Avagadro's number (where 6x1023 molecules
    1 mole)
  • We must have (6x1023) x (3x10-15) molecules
    1.8x109 copies

49
Qualitative Estimation in HIV
  • Resistance-conferring mutations identified for
    major anti-retroviral drug classes nucleoside
    reverse transcriptase inhibitors, non-nucleoside
    reverse transcriptase inhibitors and protease
    inhibitors
  • PCR gives increased sensitivity, low cost and
    high through-put.
  • Resistance testing - increasingly important in
    HIV management
  • expensive and not widely accessible.
  • MSPCR - simple and reliable for screening for
    key drug-resistance mutations in almost any
    clinic.
  • relevant especially to resource-poor areas where
    resistance remains poorly investigated.
  • Simple detection of point mutations associated
    with HIV-1 drug resistance Frater et al., Journal
    of Virological Methods 2001, 93145-156

50
Roche HIV Diagnosis
  • http//www.roche-diagnostics.com/ba_rmd/video_hiv_
    diagnosis.html

51
The Principle of MSPCR competitive reaction
  • primers anneal to WT or MT- incorporate 3'
    mismatches
  • mismatch mutation of template means the
    non-matching primer cannot anneal.
  • mutated template enhances specificity.
  • Original alignment WT primer mismatches at bases
    1 and 3. The MT primer mismatches at base 2.
  • At 1st round MT primer has incorporated its own
    base into posit 2, i.e no mismatches with the
    template, but 3 mismatches with WT primer.

52
Molecular Techniques for tracking viral
epidemics/ pandemics is crucial.
  • Use of E.g. SARS H5N1 avian flu 
  • Conventional diagnostic tools, cell culture, and
    serologic testing require from 14 days.
  • Commercially available rapid antigen tests (such
    as Directigen Flu AB Binax NOW) for H5N1 are
    rapid and simple but subtyping of viruses is not
    feasible.

53
Virus Tutorial
  • http//www-micro.msb.le.ac.uk/Tutorials/Time/Machi
    ne.html

54
Real-time amplification
  • First real-time amplification system used
    ethidium bromide and a mounted CCD camera to
    monitor PCR amplification in a closed reaction
    tube
  • Advancements in technology and software
    exploiting initial principle of monitoring
    changes in amplification signal with time.
  • Real-time PCR provides researchers and diagnostic
    laboratories with additional tools for
  • disease diagnosis
  • identification of species
  • quantifying gene expression
  • single nucleotide polymorphism (SNP) detection
  • monitoring infection loads during therapy

55
Advantages of using Real-Time PCR
  • Traditional PCR measured at end-point (plateau)
    real-time PCR at the exponential growth phase
  • Increased reporter fluorescent signal directly
    proportional to no. of amplicons generated
  • Increased dynamic range of detection
  • 1000-fold less RNA than requirement
  • No-post PCR processing due to closed system (no
    electrophoretical separation of amplified DNA)
  • Detection is capable down to a 2-fold change
  • Small amplicon size increases efficiency

56
Real Time Quantitative PCR (TaqMan PCR)
  • Abolishes need for internal controls / templates.
  • Involves
  • using a probe which binds to sequences to be
    amplified probe is labelled with
  • a) a fluophore
  • b) a quencher.
  • The fluoresence of the fluophore is much brighter
    when it is dissociated from the quencher.

57
Real Time PCR Animation
  • http//pathology2.jhu.edu/MOLEC/techniques_main.cf
    m

58
More Molecules More Fluorescence
  • The probe is broken up during PCR DNA polymerase
    has 5'-3' nuclease activity.
  • Therefore, as more amplicons are generated, the
    more probe molecules (present in excess) split,
    and the greater the fluorescent signal.

59
Typical Amplification Curve
  • The number of cycles required to achieve
    threshold fluorescence vs copy number initially
    added.

60
Standard Curve
  • a simple conversion graph.
  • So "unknown" (sample) which needs x amount of
    cycles to reach threshold fluorescence, must have
    originally had y copies.

61
  • Unbound intercalating dye not fluorescent (a) but
    increase in fluorescence on binding ds DNA (b).
  • Taqman probes cannot fluoresce when intact due to
    the proximity of the R and Q (c) produce signal
    after hydrolysis by Taq and release of R(d).
  • 2ndry structure of MGB Eclipse probes causes Q
    and R to be close no fluorescence (e) bound DNA
    probe is stabilised by minor groove binder and
    separates Q and R to allow fluorescence (f).

62
New Real-Time PCR Assay for Rapid Detection of
Methicillin- Resistant Staphylococcus aureus
Directly from Specimens Containing a Mixture of
Staphylococci Journal of Clinical Microbiology,
May 2004, p. 1875-1884, Vol. 42, No. 5A.
Huletsky,1,2 et al.
  • Example showing the FAM fluorescence detection of
    MRSA, using 10 copies of genomic DNAs purified
    from MRSA strains with MREJ types i (solid line),
    ii (dashed line with dots), iii (circles), iv
    (solid line with circles), v (solid line with
    hash marks), and vii (squares). Dashed line,
    negative control.

63
Evaluation of MRSA PCR assay using DNAs from a
variety of methicillin-susceptible and
methicillin-resistant staphylococcal strains
64
Comparison of Real-Time PCR Assays with
Fluorescent-Antibody Assays for Diagnosis of
Respiratory Virus Infections in Children
  • The PCR assays were significantly more sensitive
    than FA assays for detecting respiratory viruses,
    especially parainfluenza virus and adenovirus.
  • Use of real-time PCR to identify viral
    respiratory pathogens in children will lead to
    improved diagnosis of respiratory illness.
  • Kuypers J et al., (2006) Journal of Clinical
    Microbiology 442382-2388

65
Results of FA vs PCR
  1. No. of specimens ve for any six respiratory
    viruses by viral load (log10 RV copies/ml) among
    specimens that were -ve by FA (light columns) or
    ve by FA (dark columns).
  2. The number of log10 copies/ml for six RVs
    quantified by PCR in respiratory specimens that
    were ve (diamonds) and -ve (triangles) by FA

66
Variety of Amplification Techniques
  • developed in the mid to late 1980's.
  • PCR, ligation-mediated amplification and
    transcription-based amplification were refined
  • transcription-mediated amplification (TMA),
  • nucleic acid sequence-based amplification
    (NASBA),
  • ligase chain reaction (LCR),
  • strand displacement amplification (SDA)
  • linear linked amplification,).
  • some incorporated into clinical diagnostic assays
    (e.g. SDA for Mycobacteria and Chlamydia
    detection, NASBA for HIV-1, CMV and
    Enteroviruses, TMA for the detection of
    Mycobacteria, Neisseria and Chlamydia).

67
Nucleic acid sequence-based amplification NASBA
  • RNA (red wavy line) converted to ds DNA with a T7
    promoter using reverse transcriptase, RNaseH and
    a primer with a T7 promoter.
  • DNA used as a template by T7 RNA polymerase for
    production of multiple copies of antisense RNA
    (black wavy lines).
  • Each transcript acts as a template for production
    of additional ds DNA templates.

68
Transcription-mediated amplification (TMA)
  • DNA is amplified isothermally by RT and DNA
    polymerase alternately.
  • TMA works by a similar principle to NASBA, except
    that the assay relies on the RNaseH activity of
    the reverse transcriptase, rather than using a
    separate enzyme with RNaseH activity.
  •    1010 fold amplification in 30-45 minutes

69
Video of TMA
  • http//pathology2.jhu.edu/MOLEC/techniques_main.cf
    m

70
Ligase chain reaction
  • LCR developed shortly after PCR
  • Uses a thermostable DNA ligase and four primers,
    two adjacent forward primers and their
    complements.
  • A gap of 13 bases acts a template for ligation
    by DNA ligase.
  • Ligases specificity exploited for use in
    detection of species-specific differences for
    Plasmodium sp.
  • slow uptake of LCR due to dominance of PCR.
  • Commercial LCR kits for Mycobacterium
    tuberculosis perform well but the Chlamydia
    trachomatis kit had problems
  • technology holds promise for SNP detection and
    use in microchips or with universal microarrays

71
Strand displacement amplification (SDA)
  • no heat / cooling cycles
  • uses restriction enzyme to cleave primer
    polymerase then restarts elongation.
  • Fluorescent probe changes signal on annealing to
    amplicon (molecular beacon).

72
Improved SDA
  • The original SDA process improved by
    incorporating a thermostable polymerase and a
    different exonuclease to greatly improve the
    yield and rate of amplification.
  • 1010-fold amplification of target after 15 min at
    60 C
  • SDA can also be used to detect RNA by
    incorporating a reverse transcription step

73
Application of SDA
  • For clinical diagnosis of pathogenic organisms
    (HIV-1 M. tuberculosis, Chlamydia and Neisseria
    and pathogenic E. coli.
  • Latest paper de Silva T et al., The significance
    of low-positive MOTA scores in the
    BDProbeTec-Strand Displacement Amplification test
    for the detection of Neisseria gonorrhoeae.J
    Clin Microbiol. 2006 Oct 11 Epub ahead of
    print

74
DNA helicase allows isothermal DNA amplification
  • A combination of DNA helicase, single-stranded
    DNA-binding proteins and accessory proteins are
    used to unwind double-stranded DNA, which can
    then act as a template for DNA synthesis using
    primers and a DNA polymerase.
  • used to detect Treponema denticola and Brugia
    malayi
  • Recently helicases that display activity in the
    absence of accessory proteins identified.

75
Latest Methodologies
  • DNA chips (high density oligonucleotide probe
    arrays)
  •     - based on hybridisation technology.
  • Probes (106) are fixed to a glass chip
    fluorescently tagged sample DNA is added and
    fluorescence recorded.
  • PCR is just one example of the target
    amplification method.

76
Microarray Animation
  • http//www.bio.davidson.edu/courses/genomics/chip/
    chip.html

77
Array-based assays
  • instantaneous detection of pathogens and
    prediction of antimicrobial resistance
    revolutionise management of infection.
  • a range of characteristics to be rapidly and
    simultaneously determined.
  • As cost of DNA microarrays or 'chips' reduce
    they will be used for more routine applications.
  • Microfluidics offers the possibility of combining
    purification, amplification and detection in a
    single disposable device microarrays are
    particularly suitable for use within these
    systems.
  • Arrays will be an important tool for clinical
    diagnostics.

78
Design of microarray probes for virus
identification and detection of emerging viruses
at the genus level
  • Traditional methods detect specific single
    viruses not novel viruses
  • identifies conserved viral sequences at the genus
    level for all viral genomes available in GenBank
  • established a virus probe library.
  • genera of emerging and uncharacterized viruses
    based on hybridization of viral sequences to
    conserved probes for the existing viral genera.
  • the use of a virus identity calculation has great
    potential in the diagnosis of viral infections
  • Chou C et al., BMC Bioinformatics. (2006)
    287232.

79
Matrix-assisted laser desorption/ionisation
time-of-flight
  • MALDI-TOF mass spectrometry has been used to
    directly detect amplification products from PCR
  • Involves linking a small molecular weight
    molecule to the 5' end of a nucleotide, which is
    used as a primer in an allele specific SNP assay,
    or as a probe for pathogen detection.
  • Detection of these linkers is achieved by
    MALDI-TOF mass spectrometry (see
  • Up to 30 linkers can be used, enabling high
    throughput screening of SNPs

80
Masscode Technology
  • A MALDI-TOF-based technology using
    photo-cleavable linkers
  • Used detect of a variety of respiratory
    pathogens, including Legionella, Influenza and
    Adenovirus, and levels of detection ranged from
    100 to 5000 DNA/RNA copies depending on the
    pathogen
  • Cost is prohibitive to most labs associated with
    purchasing a mass spectrometer, and this is
    reflected in the relatively limited application
    of the technology.

81
Nanoparticles
  • http//www.john-heseltine.co.uk/medical/click_cont
    ent.html

82
Nanotechnology
  • lab-on-a-chip systems
  • miniaturise conventional and real-time
    amplification systems, rapid analysis of
    sub-microlitre volume samples
  • Many detection systems gold nanoparticles tagged
    with short segments of DNA to multicolour optical
    coding for biological
  • Co-migration electrophoregrams in combination
    with restriction enzyme digest have been used on
    chip devices for discrimination and quantitation
    of PCR products and semi-quantitation of
    SARS-coronavirus has been described (Juang et
    al., 2004).

83
Detection Process of the Diagnostic Chip
  • begins with coating antigen on the detection
    area.
  • The optimum time for antigen reaction and 2nd
    antibody reaction is 5 and 15 min, respectively.
  • The total detection time is 20 min.

84
Automatic bio-sampling chips integrated with
micro-pumps and micro-valves for disease
detection.
  • microfluidic system uses membrane-movement to
    fabricate micro-pneumatic valves/pumps to form a
    bio-sensing diagnostic chip.
  • uses smaller amounts of samples and reagent
  • Could provide a useful tool for fast disease
    detection and be crucial for a micro-total-analysi
    s system
  • detection of hepatitis C virus (HCV) and syphilis
    has been performed using the bio-sampling chips.

85
Summary
  • method dictated by sample and biological question
  • cost and ease of use including assay design and
    ease of data interpretation.
  • numerous real-time PCR instruments and numerous
    detection chemistries with advantages and
    disadvantages
  • Lab-on-a-chip devices may revolutionise medical
    management and environmental monitoring - need to
    be workable in field environments.
  • future techniques will be developed to be faster,
    cheaper, and easier to use.
  • Tools currently available offer myriad of options
    to answer specific biological questions.

86
Further Reading
  • Nucleic acid amplification-based techniques for
    pathogen detection and identification Paul T
    Monis , and Steven Giglio , Infection, Genetics
    and Evolution Volume 6, Issue 1 2006, Pages 2-12
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