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