Transcript Communicable Diseases I

Communicable Diseases I & II
Dr U L Fairbrother
Communicable Disease Agents
Are….?
• Name four
Communicable Disease Agents
Are:
•
•
•
•
Bacteria
Fungi
Protozoa
Viruses
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)
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…
Gram Stain of Bacteria:Gram
Positive
• Bacteria that up take
the original purple dye
only have a cell wall
• Eg Staphylococcus
epidermis
• Causes boils
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, gutdwelling
Gram Staining Video
• http://www-micro.msb.le.ac.uk/Video/gram.mov
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.
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).
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.
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.
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
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
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.
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
Application
Drug testing
Therapeutic
Drugs of abuse
Infectious diseases
STDs
Non STDs
Endocrinology/horm
ones
Thyroid
Non-thyroid
Immunology
Allergy
Autoimmunity
Other
Cancer
Other
Share
26%
20%
6%
Analyte type
Small molecules
21% Bacteria, viruses,
13% large molecules,
8% antibodies and
metabolites
33% Small molecules,
20% large molecules
13%
including Abs
(70% <5 kDa)
7%
Antibodies
3%
3%
1%
5% Large molecules,
antibodies
8%
Concentration range
10 nM - mM
low
pM - nM
pM - nM
pM - nM
> nM
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.
Enzyme-linked immunosorbent
assay
• It employs an enzyme label for detection of
antibody–antigen complexes formed on a
solid phase.
• Detection of antibody–antigen 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.
ELISA
Autoimmune and
infectious diseases
diagnosed using ELISA
Coeliac disease
Immune reactions to food
Infectious reactions to food
Infectious diseases such as
syphilis and TB
Lupus disease
Pernicious aneamia
Systemic rheumatic disease
Thrombosis
Thyroid disease
Renal disease
• the ELISA is one of
the most widely used
immunodiagnostic
tools, particularly in
diagnosing infectious
disease
• Gosling 1990
ELISA Animation
• www.immunospot.com/elisa-animation.html
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 0157:H7
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, methicillinresistant Staphylococcus aureus (MRSA),
vancomycin-resistant enterococci, and
fluoroquinolone-resistant (FQR) strains of gramnegative bacilli and Streptococcus pneumoniae.
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.
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.
PCR advantages:
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•
•
•
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.
Interactive Lab:Bacterial ID lab
• http://www.hhmi.org/biointeractive/disease/
vlab.html
• Split into six groups of four and do one step
per group
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.
Mechanics of RT PCR
• Reverse transcribed
with reverse
trancriptase, dNTP and
random primers
• PCR primers dNTPs
and Taq polymerase
• First strand is a
duplex.
RT PCR Animation
• http://www.bio.davidson.edu/Courses/geno
mics/RTPCR/RT_PCR.html
Characterisation for Resistance
Genotype
• by PCR and DNA sequencing.
• Identifies clonal spread in clusters of
multiresistant pathogens.
• E.g MRSA, Enterobacteriaceae, ESBLproducing 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.
PCR Based method for
MRSA
• rapid identification of methicillinresistant 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 MethicillinResistant Staphylococcus aureus (2002)
Oliveira DC, de Lencastre H. Antimicrob
Agents Chemother. 46: 2155–2161.
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
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
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.
J
• Med Microbiol 52 (2003), 229-238
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.
Restriction Enzyme Digestion of
Protozoan PCR Product
 Human type of
Cryptosporidium parvum
causes waterborne life
threatening diarrhoea, in AIDS
patients.
• Restriction fragment
length polymorphism:
– amplification of target
sequence and digestion
 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.
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
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.
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.
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.
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.
Molecular Quantitation of
Pathogens:DNA 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.
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.
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 T:C ratio.
• T: amount of amplified product from target.
• C: amount of amplified product from competitor.
• When T:C = 1, we then know amount of target
DNA.
Schematic of Quantitative
Estimation
Calculating Copy Number
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•
•
•
•
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).
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
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.
• MS–PCR - 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, 93:145-156
Roche HIV Diagnosis
• http://www.roche-diagnostics.com/ba_rmd/video_hiv_diagnosis.html
The Principle of MS–PCR:
competitive reaction
• primers anneal to WT or MTincorporate 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.
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 A+B Binax NOW)
for H5N1 are rapid and simple but
subtyping of viruses is not feasible.
Virus Tutorial
• http://www-micro.msb.le.ac.uk/Tutorials/Time/Machine.html
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
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
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.
Real Time PCR Animation
• http://pathology2.jhu.edu/MOLEC/techniques_main.cfm##
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.
Typical Amplification Curve
108 107 106 105 copies
fluorescence
"Threshold"
10
20
Cycle
number
30
•The number of
cycles required
to achieve
threshold
fluorescence vs
copy number
initially added.
Standard Curve
No. of cycles
required to
achieve
threshold (CT)
105
106
107
108
Log. copy number added
• a simple conversion
graph.
• So "unknown"
(sample) which needs
x amount of cycles to
reach threshold
fluorescence, must
have originally had y
copies.
• 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).
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.
Evaluation of MRSA PCR assay using DNAs from a variety
of methicillin-susceptible and methicillin-resistant
staphylococcal strains
No. (%) with PCR result
Staphylococcal strain type (no. of strains)a
MRSA (1,657)
MSSA (569)
MRCoNS (212)
MSCoNS (74)
Positive
1,636 (98.7)
Negative
21 (1.3)
26 (4.6)
0
0
543 (95.4)
212 (100)
74 (100)
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 44:2382-2388
Results of FA vs PCR
(A)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).
(B) The number of log10
copies/ml for six RVs
quantified by PCR in
respiratory specimens
that were +ve
(diamonds) and -ve
(triangles) by FA
Variety of Amplification
Techniques
• developed in the mid to late 1980's.
• PCR, ligation-mediated amplification and transcriptionbased 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).
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.
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
Video of TMA
• http://pathology2.jhu.edu/MOLEC/techniques_main.cfm##
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 1–3 bases acts a template for ligation by
DNA ligase.
• Ligase’s 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
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”).
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
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 BDProbeTecStrand Displacement Amplification test for the
detection of Neisseria gonorrhoeae.
J Clin Microbiol. 2006 Oct 11; [Epub ahead of
print]
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.
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.
Microarray Animation
• http://www.bio.davidson.edu/courses/genomics/chip/chip.html
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.
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) 28;7:232.
•
Matrix-assisted laser
desorption/ionisation time-offlight
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 MALDITOF mass spectrometry (see
• Up to 30 linkers can be used, enabling high
throughput screening of SNPs
Masscode Technology
• A MALDI-TOF-based technology using photocleavable 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.
Nanoparticles
• http://www.john-heseltine.co.uk/medical/click_content.html
Nanotechnology
• lab-on-a-chip systems
• miniaturise conventional and real-time
amplification systems, rapid analysis of submicrolitre 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 SARScoronavirus has been described (Juang et al., 2004).
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.
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-totalanalysis system
• detection of hepatitis C virus (HCV) and
syphilis has been performed using the biosampling chips.
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.
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