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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: • • • • 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 • • • • • 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