Presentation on theme: "Waterborne Pathogens and State-of-art Detection Methods"— Presentation transcript:
1Waterborne Pathogens and State-of-art Detection Methods Dr Bharat Patel,Associate Professor in Molecular Microbiology &Director, Clinical Microbiology PG Program,School of Biomolecular & Biomedical Sciences,Griffith University, BrisbaneAustraliaFirstly, I would like to welcome all of you to the workshop on “Waterborne pathogens and state-of-the-art detection methods” which will be presented by myself and Dr Wen-Tso Liu. I hope that will find the 2 day workshop an enjoyable learning event. I also hope that you will participate in this workshop with questions, comments and suggestions and make the workshop into a learning forum for both, you as attendees and us as speakers. Your participation will make this workshop less formal and make it more encompassing.What I would like to do is give you a brief introduction by the speakers, that is, myself and Dr Wen-Tso. I hope that we can follow this by brief introductions by all of you.
2Indicators of Water Pollution Section I.Indicators of Water Pollution
3CONTENTThe Australian Cooperative Research Centres (CRC)1.1 Concept1.2 The FiveWater Related CRC1.3 CRC Water Quality & Treatment2. Introduction2.1 Microbes on our planet & their role2.2 Water as an environment2.3 Microbes & their role in water2.4 Why monitor water supplies?2.5 Ensuring the safety of drinking water.3. Bacterial Indicators of Pollution3.1 What are Bacterial indicators of pollution3.2 Total coliforms3.3 Changes in coliform definitions4. Alternatives to Total Coliforms
4Risk Assessment Analysis Framework and Pathogens Section II.Risk Assessment Analysis Framework and Pathogens
52.The current list of pathogens CONTENT1. Epidemiological data on some pathogens.2.The current list of pathogens3.How to monitor and assess the risk of pathogens?
6SECTION III. Molecular Biology Databases and Tools
8SECTION IV.The Biology, Methods for Detection, Identification & Quantitation of Water-borne Pathogens
91. The Biomolecules & Molecular Biology of Cells CONTENT1. The Biomolecules & Molecular Biology of Cells2. Biomolecule Based Technics3. The Biology & Detection Methods of Some Pathogens4. Modern Techologiesa. Polymerase Chain Reaction (PCR)b. Real Time PCRc. Pulse Field Gel Electrophoresisd. New High Throughput Methods
10Indicators of Water Pollution Section I.Indicators of Water Pollution
111. The Australian Cooperative Research Centres (CRC)
121.1 The Concept of Cooperative Research Centres (CRC) in Australia Participation by industry, universities, CSIRO and State Government bodies$1 (in cash / in kind contributions) : $1 (cash from Fedral GovernmentCommercial focusSkill acquisition and training for the industry.Usually run by an independent board65 CRCs currently on the books ($320 million pa from participants to $140 million pa cash from Fedral Government).CRCs may have synergies:CRC Water Quality& TreatmentCRC FreshwaterecologyCRC MicroelectronicengineeringCRC WastewaterTreatment
131.2 The Five Water Related CRCs CRC for Catchment HydrologyCRC for Waste Managementand Pollution ControlCRC for Freshwater EcologyCRC for Water QualityAnd treatmentCRC for Coastal Zone, Estuariesand Waterway Management
141.3 The Cooperative Research Centre for Water Quality & Treatment 2nd Round of Funding:Participants: 12 Research organisations (8 Universities), 16 Industry & 8 Associate partnersKey Objectives:create a centre of excellence with the capability of pursuing world class research and training.ensure that participants with their differing disciplines and backgrounds will interact effectively to optimise research outcomes.increase the human skills base of the water supply industry and to train new post graduate students with specialist water quality skills.commercialise Project Intellectual Property in such a manner as to ensure that the maximum benefit accrues to the Australian water industry, the Australian environment and the Australian economy generally
162.1 Microbes on our planet & their roles 60% of the organisms are microbial (more microbes than human cells)Surive & thrive in virtually in all environments, often where no other “higher forms” of life exist.1% have been characterised (24 kingdoms) & 99% remain uncharacterised (the tree of life has been generated using rRNA as chronometers)Efficient colonisers (rapid growth & doubling)Provide a service to the planet:Ecosystem servicing (biogeochemical cycle, flux)Biotechnology (vitamins, amino acids)Also produce harm:Directly as pathogensIndirectly producing byproducts (toxins)Simple morphology provides very little clues to their identities
17Water Microbiology as it Relates to Public Health NEWWater Microbiology as it Relates to Public HealthAnimal reservoirsHuman reservoirDomestic useLand surfaceGroundwaterShellfishAerosolRecreationWastewaterSurface waterAerosolsCropsThree main routes must be considered to prevent the spread of waterborne (& foodborne) diseases. The particular pathogen, its reservoir and its mode of transmission. The figuree shows the potential route(s) of transmission and the reservoirs. For examples, cows are sources for crypotosporodiosis and poultry are sources for campylosis.
182.2 Water as a Changable Heterogeneous Environment Climate variabilityRainfallSoil erosionCatchment runoffReservoirEnvironmental flowsWater allocationIrrigationBillabong (wetland)10. Drinking water Filtration plant11. Constructed wetland12. Urban run-offs13. Wastewater treatment14. Industrial use15. Industrial Re-use16. Bore17. Water table18. River sediment19. Mangrooves20. Estuary21. Recreational use
202.3 Microbes & their role in water In nature, microbes live as communities (compete, synergy, complement)They can change the environment for their growthMost natural ecosystems are pristine ie very little nutrientsWhat about reservoirs or dams (man made to maximise storage)A case study of what goes on in a reservoir: Activities affecting a reservoir
21C, N, S, O fluxes & transformations Danger Donot enterRecreationFarming activitiesstratificationpumpForestryactivitiesC, N, S, O fluxes & transformationsFiltration & treatmentLead pipeCopper pipeDistribution systemINTERACTIVITY & INTERDEPENDENCYEcology, Environmental & Public Health Microbiology GroupsRegulatory GroupTransparency GroupPVC pipeBiofilm development ?
222.4 Why monitor water supplies? Pathogens (produce disease):Present in water due to human / animal fecal contaminationBacteria, virus, protozoa, helminthsDiverse types present (eg 100 types of viruses)Chemical pollutantsCarcinogens, toxins, endocrine disruptors & treatment byproductsPresent due to industry, microbial activities, geologicalRisk to Human HealthDose, host resistance (age, immunity), length of exposure
232.5 Ensuring the safety of drinking water (management) Primary assessment: Correct operation of water supply systemVerification: Proof that water is safe after supply. This includes monitoring for compliance.Risk assessment: Maximum Acceptable Concentration (MAC). Should be zero but rarely technically & economically feasible. Compliance parametersCompliance & risk assessment may be different for countries, states and applications.Improved awarness: Flexible, transparent, achievable & realistic outcomes
242.6 Ensuring the safety by monitoring & detection Direct measurement of harmful agentsMicrobes: Not usually undertaken. Difficult, expensive, time consuming & lack of technology. Risk -> Acute & short-livedChemicals: Usually undertaken. Technology exists. Risk -> Chronic exposure & delay between sampling, testing & acting on results is okayMonitoring water quality barriers (catchment activities, filtration, disinfection)Complete risk management system for health. Gaining popularity.Currently used indicators of water qualityInadequate, but will be used until “new” & “better” methods tried, tested & ratified.Does not take into account emerging risks (microbes, chemicals). New risks, new ways.
263.1 What are bacterial indicators of pollution? Direct pathogen identification / isolation is impractical and / or impossibleAlternate indirect “indicator organism” based inference is necessary:universally present in large nos. in warm blooded animal faecesreadily detectable by simple methodsdo not grow in natural waterspersistence in water treatment regimes is similar to that for pathogens
273.2 Coliforms & E.coli as bacterial indicators (Pre 1948) Coliforms (coli-like, 1880) fulfill these criteria as they indicate fecal pollution and therefore “unsafe water”Total coliforms (Enterobacteriaceae): Escherichia, Klebsiella, Enterobacter & Citrobacter - Ferment lactose, 1% or 109/g human faeces. Used as a standard for testing (assuming that total coliforms = E. coli)PROBLEMS WITH TOTAL COLIFORM RULEProportion of E. coli & coliforms as faeces leaves the body. (Coliforms are are normal inhabitants of unpolluted soils & water).Coliforms & waterborne disease outbreaks are not always linked & does not necessarily indicate potential health risk.The current guidelines for drinking water & freshwater recreational waters are shown in the next table as comparisons
28Maximum no of indicated organisms permitted / 100 ml of water type Table Bacteriological drinking water & recreational freshwater standards or guidelinesSource of standardMaximum no of indicated organisms permitted / 100 ml of water typeTotal coliformsaThermotolerant coliformsTurbidtyb(NTU)DrinkingRecreationalWHO1-10<1-5Canada<10200c35European Economic Community<10,000d0-4United States200e<2,000d1 (monthly)Enterococci(recreational)a < 1 out of the <40 monthly samples analysed or < 5% of the > 40 samples analysed monthly should be positive for coliformsb Nephlometric Turbidity UnitsC > 90% are E.colid Compulsory limits, bathing is restricted if >20% samples over 14 day period are positivee If 5 samples taken over 30 days are positive
293.2 Coliforms & E. coli as bacterial indicators (Post 1948) Rapid methods of identifying were E. coli developedSpecific & well known thermotolerant (faecal) coliform test developed.The Total Coliform Rule has been revised, reviewed, reassessed but not dropped (Criteria based on quality & compliance & health risk assessment)Example 1. US Envrion. Protection agency (USEPA, 1990): The water authority must not find coliforms in > 5% samples. If found, repeat samples within 24 hrs. If repeat samples test positive then it must be analysed for faecal coliforms and E. coli. A positive test signifies Maximum Coliform Limits (MCC) violation & this neccessitates rapid state and public notification.Example 2 EU Directive, 1998: E. coli, Enterococci & Coliforms 0 / 100ml. Aesthetic parameters (color, conductivity, chloride, taste & ordour). The parameters should be taken in the context of health risk assessment.
303.3 Recent changes in coliform definition Coliforms: Members of the family Enterobactericeae; produce acid & gas from lactose ( ±2oC)Thermotolerant (fecal) coliforms: As above but were able to grow & ferment lactose at 44.5±0.2oC and include E. coli < Klebsiella, Enterobacter & Citrobacter (E. coli also produce indole from tryptophan). SEE “TESTS FOR DIFFERENTIATING COLIFORMS” SLIDEReport 71, 1994 Bacteriological Examination of Drinking water supplies: biochemical definition changed to “acid-only production from lactose” & therefore increased the numbers of species in the coliform categoryEnzymes: Lactose fermentation by the presence of -galactosidase is now considered as another modification to the coliform definition.Australiasia, UK, Europe & soon USEPA use commercial enyme kits & these detect coliforms that are not traditionally picked up culture media (Noncultural but viable) hence increasing the numbers of species in the coliform group.
31Klebsiella, Enterobacter, Citrobacter Table showing coliform members by evolving definitionAcid & Gas from lactoseAcid from lactose-galactosidaseEscherichiaKlebsiella, Enterobacter, CitrobacterYersinia, Serratia, Hafnia, Pantoea, KluyveraCedeceaEdwingellaMoellerellaLeclerciaRahnellaYokenellaColiforms that can be present in the environment & in human faeces (bold ) and coliforms that are only environmental (bold & underlined)
32Commercial kits based detection methods for microbial indicatorsKit Manufacturers:IDEXX: Enterolert, Colisure, ColilertHach: m-ColiBlueBioControl: ColiCompleteChromocult: MerckGelman: MicroSureIndicator groupEnzyme / (substrate)Total Coliforms-D-galactosidase (o-nitophenyl, 6-bromo-2-napthyl, 5-bromo-4-chloro-3-indolyl linked to -D-galactoside) SEE NEXT SLIDEE. coli-D-glucoronidase (5-bromo-4-chloro-3-indolyl, 4-methylumbelliferyl linked to -D-glucoronide) SEE NEXT SLIDEEnterococci-D-glucosidase (4-methylumbelliferyl, indoxyl- linked to -D-glucoroside)
33Tests for differentiating coliforms E. coliE. aerogenesK. pneumoniaeLactose35 oCEnzyme-galactosidaseONPGTotal coliformsElevated temperature44.5 oCFecal coliforms-glucoronidaseMUGIf growth atofdesignate asall fermentatusesnameddetected withassay forTests for differentiating coliforms
344. Alternatives to Coliforms as indicators of water pollution
35Faecal coliform absence indicates enetric pathogens most likely absent but does not guarantee absence of viruses & protozoal cysts (survive longer in water & more resistant to disinfection)Enterococci, sulfite-reducing clostridia, Bacteroides fragilis, Bifidobacteria, bacteriophages & non-microbiological indicators (faecal sterols) have been proposed as alternatives to fecal coliformsEntercocci is the most preferred (also as alternative to E. coli)Common commensals in warm blooded guts19 species (faecium, faecalis, durans, hirae dominate)Survive longer & do not grow in the environmentAn order of magnitude less than coliformsCommercial test available
36Risk Assessment Analysis Framework and Emerging Pathogens Section II.Risk Assessment Analysis Framework and Emerging Pathogens
372.The current list of pathogens 1.Epidemiology of some waterborne pathogens.2.The current list of pathogens3.How to monitor and assess the risk of pathogens?
38Epidemiology of some pathogens. 1.Epidemiology of some pathogens.
391. 90% water related illness are microbial Information modified1. 90% water related illness are microbial2. Canada (1974 – 1987): 32 waterborne outbreaks - Giardia:10, Norwalk & HAV: 5, 17 unknown origin. 2000: E. coli O157, 2001 Cryptosporidium.3. USA (1993 – 1998): Cryptosporidium (Milwaukee, Las Vegas, Nevada)2001: Microcystin & cylindrospermopsin found in Florida drinking water plant (5 times WHO guideline)4. Europe (1980 –1990): Cryptosporidium (UK)6. Vibrio cholera surveillance in India: 34 k (33 deaths) Flood related since July 20015. E. coli >feces contaminated soil, to irrigation water, to food(Both E. coli 0157:H7 and VT6 gene strains isolated)Swaziland: 1992 (20k), Missouri: 1989, UK: several outbreaks reported, Wyoming: 1998, NY: 1999 (1k involved, 2 deaths, Campylobacter also implicated), Canada: 2001 (2K involved, 7 deaths- heavy rainfall & inadequate treatment)6. Northern Ireland: 2001 Cryptosporidium7. Portugal: 2001 Cyanobacteria toxins reported
408. Multiagent waterborne disease outbreaks: - Switzerland: 2001, coinfection of small round structuredvirus (SSRV) + Shigella + Campylobacter- Canada: 2001, E. coli 0157:H7 & Campylobacter -> 2300 ill, 27 developed haemolytic uraemic syndrome complications (HUS), 7 deaths.
42Waterborne Pathogens: are classified as members of domains Bacteria, Eucarya or virus.they differ in:morphologiesgrowthphysiology & metabolismfine genetic detailsBoth classification & Identification is now increasingly based on theirmolecular events & molecular details (see next figure).The pathogens listed in the following tables have been detected inwater and / or in outbreaks. An attempt has been made to providetheir classification on the newly introduced molecular trend.The biology of a number of the pathogens will be described and the possible targets sites for their identification highlighted.
43Evolution of Universal Ancestor (3.5 billion yrs) EUKARYA (7) ARCHAEA (3) BACTERIA (21)TrichomonadsCrenarchaeotaEuryarchaeotaPyrodicitumSlime moldsRed algaeFungiGreen algaeDinoflagellatesCiliatesMethanocococcusPlantsDictyoglomusDesulfurococcusThermomicrobiaHalophilesAnimalsThermococcusThermodesulfobacteriaThermotogaChrysiogenetesBrown algaeAquifexDeferribacterProteobacteriaThermalesNitrospiraFlagellatesCyanobacteriaMethanopyrusFirmicutesVerrucomicrobiaMicrosporidiaAcidobacteriaKorarchaeotaFibrobacterDiplomonadsPlanctomycetesSome 15 years ago, Woese and his colleagues provided evidence based on comparisons of rRNA sequences that all life cellular forms could be unified into one “universal tree of life”. They also showed that they were 3 cellular lines of descent radiating from the tree of life which they proposed to call domain (super kingdoms). This was different from the dichotomous concept of Procaryotes and Eucaryotes (based on EM) and also different to the 5 kingdomw classification system (based mainly on phenotypic or expressed features). Subsequently, they were able to root the tree of life, that is, showed which was the most ancient domains and which was the most modern domain. Fossil evidence and the rRNA work provided evidence that cellular life started to evolve some 3.5 billion to 4 billion years ago.The tree of life is based on molecular sequences, namely rRNA.Eucarya are the most recently evolved (300 million), the Archae the most ancient.Eucarya and Archae are sister branches at the molecular level. More closely related than Archae and Bacteria (though both are both procaryotes ie resemble each other at the EM level)The Bacteria are the most diverse, 21 kingdoms cultured & at least 15 kingdoms, inferred from 16S rRNA gene sequences have no cultured representativesThe ancestor for all cells were most likely from high temperature environments, simple biochemically (anaerobic, autotroph and no light)The difference in length of the node of one branch to another is known as the distance. The closer the distance the more related they are.NOTES: Water borne pathogens of domain Bacteria include members of the phyla (kingdoms) Proteobacteria (alpha, beta, gamma and delta subdivisions) Spirochetes, Cyanobacteria and Actinobacteria.ActinobacteriaChlamydiaEvolution of Universal Ancestor (3.5 billion yrs)FusiformsSpirochetesBacteroidesThe Tree of Life - 16th November 2000
442. A list of bacterial waterborne pathogens Bacterial pathogenPhylumFecesUrineDiseaseHASphingomonasPotentialBurkeholdariaE. coli 0157:H7 (hemorrhagic)E. coli (enteroinvasive)E. coli (enterpathogenic)E. coli (enteroitoxigenic) Enterobacterales+-Strain dependent cramps, vomit, diarrhea, feverSalmonellae speciesSalmonella enterica (serovar typhi)Watery, bloody diarrheaTyphoid, enteric fever, abdominal painShigella (S.flexneri, S. sonnei,S. dysenteriae, S. boydii)Shigellosis (bacillary dysentery)Plesiomonas shigelloides?Fish & crustaceansVibrio cholera 01Vibrio cholera non-01 VibrionalesCholera (Asiatic flu, Indian, El Tor)Legionella LegionellalesLegionellosisPseudomonas PseudomanadalesAermomonas hydrophila AeromoandalesWater diarrheaDesulfovibrio species DesulovibrionalesStomach colitis (?)CampylobacteriaDiarrheaArcobacterHelicobacteriaStomach ulcersLeptospiraSpirochaetesWeil’, swineherd’s, hemorrhagicMycobacteria avium-intracelllare& other speciesActinobacteriaLung diseaseCyanobacteria (toxins)Cyanobacteria: taxonomy in fluxMycrocystins (60), CylindrospermopsinProteobacteriaDuration of disease is between 1 to 42 days
45Problems associated with bacterial identification Information modifiedProblems associated with bacterial identificationPhylum Cyanobacteria (blue green algae):Some 50 to 60 genera; some produce oligopeptide toxins& are of increasing concern (dermal, cytotoxin, mutantion causing and carcinogens). Lifelong exposure vs short term acute exposureToxins are produced by (a) nonribosomal peptide synthetase (NRPS) which have iterative catalytic domains. Overproduction of one or several sets up a catalytic reaction leading to production of the toxins. (b) Peptide kinase synthetase (PKS).MALDI-TOF MS shows a large spectrum of oligopeptides & other poorly undertood metabolities from cyanobacteria.Microcystis exist as toxigenic organism in reservoirs & form blooms (summer to late autumn) but reports of non-toxicogenic strains have been reported.Some 60 toxins (collectively called Microcystin) are produced; these are thought to react with chlorine to produce other toxin bye-productsThey have been traditionally classified on the basis of morphology & physiology which has created confusion. Based on 16S rRNA and DNA homology studies, the 23 species have now been identified as belonging to M. aeruginosaToxin production in strains vary based on growth conditions (in vivo and in situ) causing more confusion.
46Nostoc punctiforme PCC 73102. "Anabaena cylindrica" str. NIES19 PCC 7122.Pseudoanabaena biceps PCC 7367.Lyngbya confervoides PCC 7419."Calothrix desertica" PCC 7102.Cylindrospermopsis raciborskii str. AWT205."Anabaena variabilis" IAM M-3.10%Nostoc muscorum PCC 7120.Planktothrix rubescens str. BC-Pla 9303."Oscillatoria agardhii" str. CYA 18."Oscillatoria corallinae" str. CJ1 SAG8.92.Trichodesmium speciesSpirulina subsalsa str. M-223.Prochloron didemni.Cyanobacterium stanieri PCC 7202."Oscillatoria rosea" str. M-220.Merismopedia glauca str. BGloeothece membranacea.Microcystis wesenbergii.Microcystis novacekii str. TAC20.Microcystis viridis.Microcystis ichthyoblabe str. TAC48.Microcystis aeruginosa.Chamaesiphon subglobosus PCC 7430.Octopus Spring microbial mat DNA YellowLeptolyngbya boryanum PCC"Plectonema boryanum" UTEX 485.Leptolyngbya foveolarum str. Komarek 1964/112.Gloeochaete wittrockiana str. SAG B 46.84Glaucocystis nostochinearum str. SAG 45Cyanophora paradoxa (colorless flagellate alga) -- cyanelle."Oscillatoria limnetica" str. MR1Phormidium mucicola str. M-221.The identification of cyanobacteria, the causative agents for a number of toxin-producing illnesses, is in a state of flux. The previous identification by morphology & / or toxin production does not reflect the rRNA based molecular phylogeny.Phormidium ambiguum str. M-71.Microcystis holsatica.Microcystis elabens.Prochlorococcus marinus PCC 9511.Synechococcus elongatus.Prochlorothrix hollandica."Oscillatoria neglecta" str. M-82Phormidium "ectocarpi" str. N182.Phormidium minutum str. D5.
472. A list of protozoal waterborne pathogens SourceDiseaseAnimal fecesNon-fecalEntamoeba histolyticaRareNoAmebiasis (dysentry, enetritis, colitis)Giardia lambliaYesGiardiasis (hikers disease)Cryptosporidium parvumCryptosporodiosis (cramp, vomit, fever, diarrhea)Microsporidia:EnterocytozoonSeptata?Cramp, vomit, fever, diarrheaCyclosporacayatenensisToxoplasma gondiiAcanthamoebaBlantidium coliAbdominal pain, bloody diarrhea
48Picorna, Corona, parvo, picobirna, picotrirna 2. A List of viral waterborne pathogensVirusGroupFaecal SourceDiseaseHumanAnimalCytopathogenic human orphan (ECHO),polio,coxsackiesEnteroYesNoAseptic meningitis, infantile diarrhea, polioHepatitis A Virus (HAV)Hepatitis E Virus (HEV)HepatitisPigs ?Infectious HepatitisRotavirus ARotavirus BRotavirusAcute gastroenteritisNowalk virusSnow mountainCalicivirus?Astrovirus100’s of others (Developing new method to work with them?) Small small round structured virus (SSRV)Picorna, Corona, parvo, picobirna, picotrirnaUncertain
49Viruses:Role of some human enteric & respiratory viruses (& some animal viruses) as waterborne pathogens has been well establishedMost are nonenveloped (except corona & picobirna-viruses) – more ressistant to physical & chemical agents then the lipid containing enveloped virusesPotential transmission route directly or indirectly from animal human & this is of concern
50By using risk assessment analysis frame work 3. How to prioritise the list of pathogens for further studies?By using risk assessment analysis frame work
51Table 2 Ecology / occurrence framework for waterborne pathogens Occurrence determinants:Incidence,Lifecycle(s),Epidemiological data – worldwide,reservoirs of agents (animal / human),geological distributionDetection:General,viable?,temperature (water pollution)Water-based vs water borne:Secondary hostsBiofilmTreatment barriers:Source water qualityWatershed managementTreatment process configuration (driven by source water quality)Distribution concernsMicrobial adaptation:Treatment chainDistribution systemPathways:IngestionDermalInhalation
53Conclusions from discussion on pathogens Many pathogens cause water-borne diseasesComplex habitats for their growthPathogenic bacteria, virus & protozoa may co-existSymptoms similar but causative agents may be different.Thereforeassisted diagnosis is not always possibleIdentification essential as patient treatment regimes depend on thetype of causative agent (bacteria vs virus vs protozoa)Alternative methods to assess the risk of the pathogens present inwater are necessary which can be achieved by using variousframeworks
54The Need for Molecular methods for the identification & detection of pathogensCurrent US$380 million market & a 20% annual increase is expectedEmerging sophisticated gene technologies (indicators & pathogens)Skilled (bioinformatics, genomics, phenomics) staff required.Multicomprehension (ecology, environmental etc) requiredMethod rapid flexible, reproducible & can be ariticulated to particularneeds of different countriesInitial research & development outlay is expensive (research costs)
55Next? Finding molecular biology “information libraries” Understanding the principles of molecular biologyFinding & using tools for molecular methods
56SECTION III. Molecular Biology Databases and Tools
58Molecular Biology DataBases Biologists have been very successful in finding DNA & protein sequences:- high-speed automated DNA sequencing equipme- the Microbial Genomes (and eucaryotic genomes- bulk sequences of cDNAs (ESTs) especially for eucaryotic genomes.Why?- Bioinformatics scientists collect, organize and make sequence data that is generated, available to all biologists- Today data is shared and integrated between the three major data depositories, namely, GenBank, which forms part of the NCBI, European Molecular Biology Laboratory (EMBL) and the DNA Database of Japan (DDBJ).- During Oct. 1996, GenBank contained 1,021,211 sequence records = 652,000,000 bases of DNA sequence = 3.1 gigabytes of computer storage space. In June 1997 this escalated to 1,491,000 records and 967,000,000 bases. Check the sequence record out for for 2000- The contents of GenBank are now doubling in less than a year, and the doubling rate is accelerating ie the data generated and collected is growing exponentially.- Whole genome data has been generated with 32 microbial genomes sequenced. A list of completely sequenced genomes and ongoing genome projects are maintained at Genomes Online Database (GOLD).
59- Even simple computation or searching these enormous database requires a huge amount of computer power. What will be needed in 5 to 10 years time is hard to image.B. The Resources at NCBIEstablished in 1988 as a national resource for molecular biology information. It creates public databases, conducts research in computational biology, develops software tools for analyzing genome data, and disseminates biomedical information - all for the better understanding of molecular processes affecting human health and disease.The NCBI can be summarised as having 3 arms:GenBank Data Base: The GenBank Database is a sequence database and has a collecti on of publically available sequence data. It is part of National Institute of He alth (NIH), USA. GenBank, DataBank of Japan (DDBJ) and European Molecular Biolog y Laboratory (EMBL) have formed the International Nucleotide Sequence Database C ollaboration project under which the 3 organisations exchange data on a daily basis.In this database, new protein and nucleic acids sequences are deposited by researchers. These sequences are annotated and placed in the sequence database for access and public viewing. The database can also be searched.
60Literature Data Base: This is refered to as PubMed Literature Data Base: This is refered to as PubMed. The database holds the abstracte of published articles.The various Sequence Data Bases and PubMed literature Data Base are linked via ENTREZ. ENTREZ is at the core of the search and retrieval system that integrates and links th e various databases. In order to maximise the benfits of the various databases it is imperative that you read and learn from the ENTREZHELP FILEBioinformatics Tools: The most commonly used tool is known as BLAST and enables the user to input a sequence and search for the most similar sequences in the Data Base.C The Ribosomal DataBase Project (RDP):Contains downloadable GenBank formatted aligned and unaligned small subunit ribosomal rRNA sequences. Mainly extracted from the GenBank Data Base - is a GenBank subset specialist Data Base. It also conatins a set of integrated online analysis bioinformatics tools useful for aligning user input sequences based on rRNA secondary structural constraints and for constructing phylogeny.D. KEGG Data Base:
61Some Useful Online Molecular Biology Tools Search launchers atComputational Biology at EMBL:National Centre for Genome Research:UC Sac Diego Motif Search & alignment tools:The tools at InfoBiogen, France:The tools at the University of Pennsylvania:Compilation of tools & references at the University of California, Santa Cruz:
62Why study microbial genomes? until whole genome analysis became viable, life sciences have been based on a reductionist principle – dissecting cell and systems into fundamental components for further studystudies on whole genomes and whole genome sequences in particular give us a complete genomic blueprint for an organismwe can now begin to examine how all of these parts operate cooperatively to influence the activities and behavior of an entire organism – a complete understanding of the biology of an organismmicrobes provide an excellent starting point for studies of this type as they have a relatively simple genomic structure compared to higher, multicellular organismsstudies on microbial genomes may provide crucial starting points for the understanding of the genomics of higher organisms
63analysis of whole microbial genomes also provides insight into microbial evolution and diversity beyond single protein or gene phylogeniesin practical terms analysis of whole microbial genomes is also a powerful tool in identifying new applications in for biotechnology and new approaches to the treatment and control of pathogenic organisms
64History of microbial genome sequencing first complete genome to be sequenced was bacteriophage X bpfirst genome to be sequenced using random DNA fragments - Bacteriophage bpmitochondrial (187 kb) and chloroplast (121 kb) genomes of Marchantia polymorpha sequencedearly 90’s - cytomegalovirus (229 kb) and Vaccinia (192 kb) genomes sequencedfirst complete genome sequence from a free living organism - Haemophilus influenzae (1.83 Mb)late 1990’s - many additional microbial genomes sequenced including Archaea (Methanococcus jannaschii ) and Eukaryotes (Saccharomyces cerevisiae )
65Microbial genomes sequenced to date currently there are 32 complete, published microbial genomes – 25 domain Bacteria, 5 Domain Archaea, 1 domain Eukarya (www.tigr.org)around 130 additional microbial genome and chromosome sequencing projects underway
66Laboratory tools for studying whole genomes conventional techniques for analysing DNA are designed for the analysis of small regions of whole genomes such as individual genes or operonsmany of the techniques used to study whole genomes are conventional molecular biology techniques adapted to operate effectively with DNA in a much larger size range. An example is that of pulsed field gel electrophoresis (PFGE), the principle of which will be discussed in detail under Molecular Methods section.PFGE is utilised routinely for epidemiological studies and for fingerprinting of E. coli and Neisseria meningitidis genomes. A potential useful tool for studying species, strain and serovariants
67Characteristics of sequenced genomes the 32 complete genome sequences currently available cover a diverse range in terms of phylogeny and environments (eg. human pathogens, plant pathogens, extremophiles etc.)what conclusions can be made by comparing the genomes of these organisms regarding specific adaptations to proliferation in remarkably different environments?What conclusions can be made about evolutionary relationships between these organisms?
68Horizontal gene transfer before microbial genome sequences became available most of the focus of microbial evolution was on ‘vertical’ transmission of genetic information – mutation recombination and rearrangement within the clonal lineage of a single microbial populationgenome sequences have demonstrated that horizontal transfer of genes (between different types of organisms) are widespread and may occur between phylogentically diverse organismsgenerally speaking, essential genes (such as 16S rRNA) are unlikely to be transferred because the potential host most likely already contains genes of this type that have co-evolved with the rest of its cellular machinery and and cannot be displacedgenes encoding non-essential cellular processes of potential benefit to other organisms are far more likely to be transferred (eg. those involved in catabolic processes)clearly, lateral transfer of genomic information has enormous potential in improving an microorganisms ability to compete effectively - this may explain why horizontally transferred genes appear so frequently and ubiquitously in microbial genomesan example of this is horizontally transferred genes has been found in pathogenic microbes
69Whole genome phylogenetic analysis most of the evolutionary relationships between microorganisms are inferred by comparison of single genes – usually 16s rRNA genesalthough extremely effective, single gene phylogenetic trees only provide limited information which can make determining broad relationships between major groups difficultphylogenetic relationships can be determined by whole genome comparisons of the observed absence or presence of protein encoding gene familiesin effect this is similar to using the distribution of morphological characteristics to determine phylogeny – without the problem of convergent evolutiontrees produced using this method are similar to 16s rRNA trees, however, as more genome sequences become available more detailed conclusions can be drawn using this method
70Species and strain specific genetic diversity although genome sequencing and analysis is very useful when comparing phylogenetically distant taxa, it is also of interest to examine the genomes of very closely related microorganismsthis allows a more quantitative approach for examining the relationships between genotype and phenotypecomplete genome sequences have been determined for two species of the genus Chlamydia (pneumoniae and trachomatis)although the overall genome structure was quite similar, C.pneumoniae contained an additional 214 genes most of which have an unknown functiontwo strains of the bacterium Helicobacter pylori have been completely sequenced (26695 and J99)overall the two strains were very similar genetically with only 6% of genes being specific to each strain
71Case study - Neisseria meningitits N. meningititis causes bacterial meningitis and is therefore an important pathogengenome is 2.2 megabases in size2121 ORF’s were identified with many having extremely variable G+C% (recently acquired genes)many of these recently acquired genes are identified as cell surface proteinsthere is a remarkable abundance and diversity of repetitive DNA sequencesnearly 700 neisserial intergenic mosaic elements (NIME’s) - 50 to 150 bp repeat elementsthese repeat elements may be involved in enhancing recombinase specific horizontal gene transfer
72Case study - Borellia burgdorferi B. burgdorferi is a spirochaete which causes Lyme diseaseit has a 0.91 megabase linear genome and at least 17 linear and circular plasmids which total 0.53 megabases853 predicted ORF’s identified - these encode a basic set of proteins for DNA replication, transcription, translation and energy metabolismno genes encoding proteins involved in cellular biosynthetic reactions were identified - appears to have evolved via gene loss from a more metabolically competent precursorthere is significant amount of genetic redundancy in the plasmid sequences although a biological role has not been determinedit is possible the these plasmids undergo frequent homologous recombination in order to generate antigenic variation in surface proteins
73What can we learn from microbial genomes? Comparative Genomics: Multiple Pathogenecity Associated Islands (PAI) of 4 uropathogenic E.coli strains against the backdrop of E. coli strain K-12. The PAIs of 25 to 190 k, are inserted within or adjacent to tRNA genes & contain a different % GC content to the genomic DNA. Transfer mechanism(s)?pheR~25 kbselC70 kbpheV>170 kbthrwE. coli K-12Chromosome94 min97 min64 min27 min5.6 min44 min536Strain #CFT073J9653582 minleuX190 kbmetV60 kbasnT45 kb
74What can we learn from microbial genomes? Another case study of a microbial genome
75SummaryMicrobial genome sequencing and analysis is a rapidly expanding and increasingly important strand of microbiologyimportant information about the specific adaptations and evolution of an organism can be determined from genome sequencinghowever, genome sequencing merely a strong starting point on road to completely understanding the biology of microorganismsfurther characterisation of ORF’s of unknown function, in combination with gene expression analysis and proteomics is required
76SECTION IV.The Biology, Methods for Detection, Identification & Quantitation of Water-borne Pathogens
771. The Biomolecules & Molecular Biology of Cells CONTENT1. The Biomolecules & Molecular Biology of Cells2. Biomolecule Based Technics3. The Biology & Detection Methods of Some Pathogens4. Modern Techologiesa. Polymerase Chain Reaction (PCR)b. Real Time PCRc. Pulse Field Gel Electrophoresisd. New High Throughput Methods
781. The biomolecules & molecular biology of cells
79DNA RNA PROTEINS TOTAL DNA: Mol%G+C rRNA sequencing Restriction Patterns (RFLP, PFGE)Genome sizeDNA homologyrRNA sequencingLMW RNA profiles23S16SDNA SEGMENTS:PCR based fingerprinting (ribotyping, ARDDRA, RAPD, AFLP, AP-PCR, rep-PCR)DNA probesDNA sequencing5SPlasmid DNAtRNADNAElectrophoretic patterns of total cellular or cell envelope proteins (1D or 2D)Multienzyme patterns (multilocus enzyme electrophoresis)PROTEINSmRNACHEMOTAXONOMIC MARKERSEXPRESSED FEATURESCellular fatty acids (FAME)Mycolic acidsPolar lipidsQuinonesPolyaminesCell wall compoundsExopolysaccharidesMorphologyPhysiology (Biolog, API, …)Enzymololgy (APIzyme)Serology (monoclonal, polyclonal)DIFFERENT TARGETS FOR MICROBIAL IDENTIFICATION
80Selection of Different Targets NewSelection of Different TargetsCell surface:proteins (receptors, porins, siderophores): 200,000 / cellPolysaccharides (LPS): 2 million in Gram –ve cellsCytoplasmic:Ribosomes (rproteins & rRNA): 20,000 in dividing cells.Non-ribosomal RNA: 100 – 1,000 / cell (depending on rate of transcription or rate of degradation)Non-iobosomal proteins (RNA polymerase): 3,000 / cellThe target concentrations in a 1 ml sample will be 0.03 attomolar(3,000 molecules / cell) to 20 attomolar (2 million / cell)
82TechniqueFamilyGenusSpeciesStrainRestriction Fragment Length Polymorphism (RFLP)Low frequency restriction fragment analysis (PFGE)Phage and bacteriocin typingSerological techniquesRibotypingDNA amplification (AFLP, AP-PCR, RAPD)Zymograms (multilocus enzymes)Total cellular protein electrophoretic patternsDNA homologyMol% G+CDNA amplification (ARDRA)tDNA-PCRChemotaxonomic markersCellular fatty acid fingerprinting (FAME)rDNA / rRNA sequencingDNA probesDNA sequencingHighthrougput assays (Microarrays, Cantilever arrays)The limits of resolution of various techniques in microbial identification
833. The biology & detection methods of some pathogens
84Virulence Factors (VF) of Water-borne Pathogens VF encoded by genestheir presence makes the microbe pathogenicMost E. coli in human/animals not pathogenic as VF genes are absentAquatic environment may be reservoir where “virulence breed” byPlasmids/phage transmissision of VF (E. coli, Y.eneterocolitica & A. hydrophila)Viruses:Virus multiplicationMost non-enveloped. Antigenic shift & drift in capsid proteinsBacteria:Salmonella – O (in LPS, endotoxin) & Vi (capsule) antigensE. coli may contain > 1 VFs:-EIEC enteroinvasive: Shiga-like toxin (SLT),-ETEC enterotoxigenic: Vibrio like heat labile/stable toxin (ST, LT), ID > 1million cells. Interfere with Na & Cl across CM, travelers diarrhea.-EPEC, enteropathogenic: Adhesive VF for GI epithelia., infantile diarrhea indeveloping countries-EHEC, enterohemorrhagic: Shiga-like toxin (SLT), ID < 1000 cells, Since1982, strain O157:H7 has affected 20,000 in US (>100 deaths), Found in groundbeef & now in cider & fruit juices.Vibrio cholera: Cholera txin resides on plasmids which are transferred by phage
85Detection in water supplies is a challenge Protozoal Parasites:Detection in water supplies is a challengeBiology remains unstudied, biomarkers unavailableMethods have limitation & cannot differentiate:human species form animal speciesinfectious forms from noninfectious formsTechniques such as Microscopy, PCR & RFLP of limited use for diagnosticsCharacteristics:Entamoeba histolytica: a long history as a waterborne pathogen (no US majoroutbreaks reported for decades, no major nonhuman reservoir)Cryptosporidium parvum: Major problem.Microsporidia: Ubiquitous parasite of insects, human & animals. Significanceunknown.
86Diagnostic Methods 1. Recovery and Concentration: To increase pathogen concentration by physical, chemical or enrichments.2. Purification & Separation:Methods use knowledge of pathogen size, shape, density etc surface properties (hydrophilicity, reactivity, receptors), growth stages (spores, capsules, ooocytes) for this.3. Assay & Characterisation:Differentiate pathogens from all others: Qualitative / quantitative, viable / nonviable. Cultural, immunological and NA based [ NA amplification (PCR), NA identification & characterisation methods (hybridisation by gene probes, RFLP & nucleotide sequencing)]. NA based methods are specific & sensitive but incapable of differentiating live but inactivated cells from dead / noninfectious ones.
922. What is PCR?DNA replication in a tube (in vitro). Xeroxing (copying) of DNA.3. The Components of PCRThe basic components of a PCR reaction are- one or more molecules of target DNAtwo oligonucleotide primers - thermostable DNA polymerase - dNTPs4. The Process of PCREach PCR cycle requires three temperature steps to complete a round of DNA synthesis:
93Cycle 1: The original DNA template will continue to be copied by the DNA polymerase until it stops or the process is interrupted by the start of the next cycle.Cycle 2: Amplicons of intermediate lengths producedCycle 3: Amplicons of defined lengths will be produced.Cycle 4 onwards: Target sequence will be amplified exponentiallyThe final number of copies of the target sequences is expressed as: (2n-2n)xwhere n = no of cycles,2n = 1st product obtained after cycle 1 & 2nd products obtained after cycle 2 with undefined length andx= no. of copies of the original template
94PCR target molecules accumulate as a function of cycle number with the exponential phase lasting for about 30 cycles under standard reactions conditions. The plateau phase results from limiting amounts of enzyme and reduced enzyme activity. The production of 1 billion copies of the specific targeted DNA from 1 template during the 30 cycles is theoretically possible but never practically achieved because of lack of 100% PCR efficiency. The products formed during the process could be mixtures of specific and non-specific products and these factors reduce PCR efficiency from the theoretical 100%.
955. The Factors Affecting PCR I. Generalities:pipette water first, followed by the other ingredients.work “on ice” in order to minimise primers binding to the DNA template and to prevent functioning of the polymerase (even theoretically) prior to the first denaturing step.avoid aerosols while pipetting (or use aerosol-ressistant pipette tips) & work under laminar flow hoods.Be very accurate when dealing with small volumes. (Multiplex PCR of two different genomic DNA samples can be very susceptible to errors in pipetting).II Thermocyclers and PCR vials:The same PCR program will work slightly different on different thermocyclers (temperature and time profiles may differ) and therefore the PCR results using the same primer pair may vary.New PCR machine designs accommodate thin-walled 0.2 ml PCR vials (and/or 96 wells microtiter dishes). Contact between the metal and plastic is very good and aided by the downward pressure from the heated lid.Older machines accommodated 0.5 or 1.5 ml vials and the contact between the vials and the metal block is not always perfect because of slight differences in shape and wall thickness amongst manufacturers, often resulting in reduced or no amplification
96II. Denaturing temperature and time: 150mM NaCl decreases the melting (denaturing) temperatures of 91-97oC (not 100 oC)Taq polymerase has a half-life of 30 min at 95oC. Denaturation temp as short as possible2-5 minutes initial denaturing step prior to the start of cycling is not necessary1 min at 94oC (usually between 15 sec to 30 secs) avoids loss of Taq enzyme activityIII. Choosing and Primer Design:17-28 bases. Longer primers bp for multiplexingboth primers have a close melting temperature or Tm of within 5 oC.G+C content of 40-60% (Tms between 55-80oC are preferred).primer sequence with 1-2 GC pairs at the start and end improves priming efficiencythree or more Cs or Gs at the 3'-ends of primers may promote misprimingCheck for primer-primer interactions: 3'-ends of primers not complimentaryCheck for primer self-complementarity (ability to form 2o structures such as hairpins)primer sequences checked against DNA database (using BLAST programs) for “uniqueness”Useful ON-LINE programs for primer design can be found at the following URLsPrimer0.5:WebPrimer:http://genome-www2.stanford.edu/cgi-bin/SGD/web-primer Primer3:IV. Primer Annealing Temperature:Calculate Tm of primer using “rule of thumb” 4(G + C) + 2(A + T)oC.Temperature of annealing (Ta) should be about < 5oC below the lowest Tm of the pair of primersV. Extension or Polymerisation:Taq polymerase incorporates about 2000 nucleotides/minute at optimal temperature 72-78o CRule of thumb “1 min for a 1 kb product, 2 min for a two kb”
97Properties of some thermostable DNA polymerases: Some Commercial thermostable DNA polymerases and their sources:Deep Vent (Pyrococcus GB-D) RecombinantVent (Thermococcus litoralis) RecombinantUlTma (Thermotoga maritime) RecombinantTth (Thermus thermophilus) RecombinantAmplitaq (Thermus aquaticus) RecombinantAmplitaq Stoffel (Thermus aquaticus) RecombinantHot Tub – (Thermus flavus) NaturalPyrostase (Thermus flavus) NaturalTbr (Thermus brockianus) NaturalTfl (Thermus flavus) NaturalPfu – (Pyrococcus furiosus) NaturalPwo – (Pyrococcus wosei) NaturalProperties of some thermostable DNA polymerases:
98VII. Primer Amount in PCR: VI. Reaction Volumes:thin walled, 0.2 ml plastic vials for 96 well thermocyclers have heated lids (no oil). Reaction volumes (5, 25 or 100 ul) okayPCR product yield is higher in 5 µL compared to 100 µL volumes (product can be visualised)VII. Primer Amount in PCR:nM each primerPurchased as mM; 0.5-1ml primer is sufficient for ml PCR reactionsVIII. Template concentrations:Within limits, increasing primer and template concentration may improve the outcome of the PCR reaction, and should be considered as a way to optimize PCR reactionsIX. Nucleotides (dNTP):Stock of 25 mM each stored as small aliquots (2-5 µl) at -20o C.Centrifuge long term stored solutions as water condenses on the walls changing concDilute stocks in buffered water (10mM Tris pH ) as acid pH hydrolysis dNTP to dNDP and dNMPX. Relationship between MgCl2 and dNTP concentration:200µM dNTP each and 1.5mM MgCl2 is recommended with Taq polymerase, (Perkin Elmer Cetus). Theoretically µg of DNA is synthesised from 25 µl reaction. Besides magnesium bound by the dNTP and the DNA, Taq polymerase requires free magnesium. This is probably the reason why small increases in the dNTP concentrations can rapidly inhibit the PCR reaction (Mg gets "trapped") whereas increases in magnesium concentration often have positive effects.
99Real Time detection of PCR products XI. Adjuvants in PCR Reactions:Between 5-10% DMSO or glycerol promotes increase in PCR yield0.8µg/µl BSA promotes better yield than DMSO or glycerolGel electrophoresis for detecting PCR productsAgarose Gels:NuSieve agarose separates short products better than the regular agarose. More expensive but use less for the same gel strength as regular agarose.All regular agarose, irrespective of the brand, behave the same600 bp separation: Run very fast (3-4 h for a cm long 2-3% agarose gel). Bands are sharperNon-denaturing PAA gels:6-10% PAA gels used for PCR products differing in only a few bp in lengthDenaturing PAA gels:6% PAA/7M urea sequencing gel is used to separate radiolabeled multiplex PCR productsReal Time detection of PCR productsNo gels required. Recent method. Relies on the ability of a dye, SYBR Green, to intercalate with double stranded amplicons produced during PCR, to produce fluorescence which is detected in a flurometer. (Dealt with in a subsequent section)
100Distinguishing between PCR & Real Time PCR NewDistinguishing between PCR & Real Time PCRPCRPrimer design & annealingPCR cycling parameters30 cyclesDetection by Gel electrophoresisRealTime-PCRPrimer Design & annealingProbe Design internal to PCR amplicons & AnnealingPCR Cycling Parameters30 cyclesDetection of fluorescence every cycle (annealing and / or extension)Subsequent Gel Electrophoresis (if necessary)
102Real Time PCR Introduction General Principles & Concepts A. What are Fluorescent dyes?B. What is Fluorescence Resonance Energy Transfer (FRET)?C. Some commonly used flurophores for labeling probesD. Quantitating FluorescenceE. Improving Fluorescence Signal Detection (new)3. InstrumentsLightCycler (Idaho Technologies Roche)B. Rotor-Gene (Corbett Research)C. iCycler (BioRad)D. Mx4000™ Multiplex Quantitative PCR System (Stratagene)E. ABI Prism 7700 (Perkin-Elmer-Applied-Biosystem)F. SmartCycler (Cephid)
105What is Real Time PCR?Real Time PCR is a technique in which fluoroprobes bind to specific target regions of amplicons to produce fluorescence during PCR. The fluorescence, measured in Real Time, is detected in a PCR cycler with an inbuilt filter flurometer.
106WHAT IS THE DIFFERENCE BETWEEN PCR AND REALTIME PCR ? Fluorescence is measured every cycleThe signal is proportional to the amountof productObserved in Real Time during PCR
107WHAT CAN BE DONE WITH Real Time PCR? Specific quantification of: DNARNAProtein
1092A. What are Fluorescent dyes? New2A. What are Fluorescent dyes?When a population of fluorochrome molecules is excited by light of an appropriate wavelength, fluorescent light is emitted. The light intensity can be measured using a flurometer or by measuring a pixel-by-pixel digital image of the sample. In the later case, image analysis software, makes it possible to view, measure, render, and quantitate the resulting image.Excitation and Emission: Fluorodyes absorb light at one ê level (wavelength) & thereby boosts an electron to a higher energy shell (an unstable, excited state).The excited electron falls back to the ground state and the flurophore re-emits light but at a second lower ê, longer wavelength.This shift makes it possible to separate excitation light from emission light with the use of optical filters.The wavelength (nm) where photon energy is most efficiently captured is defined as the Absorbancemax & the wavelength (nm) where light is most efficiently released is defined as the Emissionmax.The difference in absorbed & emitted wavelength = Stoke’s shift (). can be a large or small number depending on the loss of energy during fluorescence process.
1102A. What are Fluorescent dyes?(cont’d) New2A. What are Fluorescent dyes?(cont’d)The wavelegth range for which flurodyes absorb light is small (~ < 50nm) and light outside this range will not cause the molecule to fluoresce.2. Linearity: Theintensity of the emitted fluorescent light is a linear function of the amount of fluorochrome present when the illuminating light has a constant wavelength and intensity (for example, using a controlled laser light source). The signal becomes nonlinear at very high fluorochrome concentrations.3. Brightness: Fluorochromes differ in how much intensity they are capable of producing. This is important because a dull fluorochrome is a less sensitive probe than a bright fluorochrome. The brightness depends on two properties of the fluorochrome-Its ability to absorb light (extinction coefficient).The efficiency with which it converts absorbed light into emitted fluorescent light (quantum efficiency).4. Environmental factors: Environmental conditions can affect the brightness or the wavelength of the absorption or emission peaks. Such fluorochromes are useful for analyzing changes in H+, Mg2+, or Ca2+ concentration & detecting lipids or double-stranded DNA. Photodestruction (photobleaching) of photosensitive dyes (eg fluorescein) is caused by intense light. Use antifade agents or lower the laser power
111Fluorescent dyes have become the preferred method of detection for nucleic acids in Molecular Biology.They are used as single conjugated dyes to oligonucleotides for:Automated fluorescent DNA sequencing,Fluorescent genotyping & Terminal Fragment Restriction Length Polymorphism (TFRLF)ANDAs double or multiple conjugated dyes to oligonucleotides for simultaneous detection, identification and quantitative techniques in Real Time PCR (Molecular Beacons) based on the principle of Fluorescence Resonance Energy Transfer (FRET) or quenching.
1122B. What is Fluorescence Resonance Energy Transfer (FRET)? Modified2B. What is Fluorescence Resonance Energy Transfer (FRET)?FRET is a distance dependent interaction interaction between the excited states of 2 dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon
1132B. FRET (cont’d):The Donor and Acceptor in close physical proximity ( Angstrom) can lead to FRET or Quenchinghv(a) Physical proximity + hvDA(FRET +ve)hvDADA(b) No physical proximity + hv(c) No hvHybridization probeshvRhvQRQ(d) Physical proximity + hv(e) No Physical proximity + hv(Quenching)(Quenching released)TaqMan & Beacon Probes
1142C. Some Commonly used flurophores for labeling probes FLUORESCEINTAMARACY 5494 / 518 nm650 / 690 nmTETCY 3HEXTEXAS REDLC-RED595 / 615 nmConsider the cost, ease of synthesis, proprietary, delivery time etc
1152D. Quantitating Fluorescence A flurometer exploits the principles of fluorescence to quantitate fluorescent (dye) molecules in the following way:A strong light source which produces light within a specific light range ( eg xenon arc lamp) is focused down to a tight beam.The tight beam of light is sent through a filter which removes most of the light outside of the target wavelength range for a particular fluorescent molecule.The filtered light beam passes through the liquid target sample striking some of the fluorescent molecules in the sample.Light emitted from the fluorescent molecules that is traveling orthogonal to the excitation light beam pass through a secondary filter that removes most of the light outside of the target wavelength range.
116The filtered light then strikes a photodetector or photomultiplier which allows the instrument to give a relative measurement of the intensity of the emitted light.Fluorescent molecules can be detected at concentrations below a level visible to the subjective human eye & as fluorescence intensity vs. concentration is a linear relationship, dye concentrations can be determined with a good degree of accuracy
1172E. Improving Fluorescence Signal Detection A number of ways are available to improve detection and measurement of the emitted fluorescent signal.a. Elimination of the excited light from the collection pathway by several methods:Orienting the excitation light path so that the light does not shine into the collection pathway.Inserting optical filters into the collection pathway to reject the excitation wavelength.Delaying collection until after a pulse of excitation light has disappeared.b. The fluorescent signal can also be enhanced by increasing the dwell time or by scanning the sample multiple times and mathematically processing the signals to reduce random noise. Such methods are useful and practical for increasing the sensitivity at the low end.c. A band-pass optical filter can be used to reject broad-spectrum background emissions. This type of filter rejects wavelengths shorter and longer than the selected band, while allowing wavelengths in the selected wavelength range (centered around the fluorescent emissions of the sample) to pass through to the collection pathway.
1182F. Advantages of Fluorescence (New) Wide variety: Fluorochromes with a wide variety of characteristics are available, including fluorochromes that -Respond to pH or ion concentrations.Localize based on hydrophobic and hydrophilic interactions.Can be cross-linked to proteins, NA, lipids, or polysaccharides.Commercial available: Fluorochromes are available crosslinked to many other molecules (eg fluorescently labeled monoclonal and polyclonal antibodies with a choice of fluorochrome, fluorescently labeled enzyme substrates, such as fluorescent chloramphenicol for chloramphenicol acetyl transferase (CAT) assays and fluorescein digalactoside for b-galactosidase assays (lacZ gene).Multiple-label possibility: A significant advantage of fluorescent labeling over other methods is the possibility of recording the fluorescence of two or more fluorochromes separately using optical filters and a fluorochrome separation algorithm. Thus, components can be labeled specifically and identified separately in the same sample or lane (EG Real Time PCR applications)
1192F. Advantages of Fluorescence (cont’d) (New) Stability: The long shelf life compared to radiolabeled molecules. Fluoromonoclonal antibodies, oligonucleotide hybridization probes, and PCR primers can be stored for six months or more but antibodies labeled with 125I and 32P-labeled nucleotides and oligonucleotides become unusable in a month and a week respectively. Reagent batches can be standardized and used for extended periods in antigen localization, ELISAs, enzyme assays (such as CAT and kinase), PCR-based genetic typing assays (such as STR analyses), DNA sizing and quantitation, DNA sequencing, protein sizing and quantitation.Low hazard: Most fluorochromes are easy to handle, however, proper care should be observed (eg gloves) with DNA and RNA stains (mutagenic as they bind to these molecules). In contrast, lead or acrylic shields are required for handling radioactive materials and require special disposal protocols (eg shielded storage, long-term decay, or regulated land-fill disposal)Lower cost: The long shelf life and cheaper transportation and disposal costs for fluorochromes make fluorescent labeling, in many cases, less expensive than radiolabeling.
120Real Time PCR Instruments 3.Real Time PCR Instruments
121General Description of Instruments PCR cycler:96 well format, 8 tube format, capillary (glass)Air or block heaterTemeperature ramp, temperature gradientFluorescence emission & detection :FluorometerCCD cameraExcitation source: xenon, halogen, laserFluorescent Dye Labeling of:OligonucleotidesPeptide Nucleic acids (PNA)Near Infra Red Dyes:Available but no commercial labeling service available
122Idaho LightCycler Xenon Arc lamp (250-1000 continuous) Glass capillaries + Air (not metal block) = rapidIdaho LightCycler
123The Lightcycler performs PCR in small-volume glass capillary tubes, contained within a rotor-like carousel, that are heated and cooled in an airstream. The carousel is rotated past a blue light-emitting diode, and fluorescence is read by three photodetection diodes with different wavelength filters that allow the use of spectrally distinct fluorescent probes. Assays based on DNA-binding dyes, hydrolysis probes, molecular beacons and dual hybridisation probes are possible. Up to 32 reactions are typically carried out in 5–20 µl volumes and PCR is completed in less than 20 min. The fluorescence readings taken at every cycle of the PCR reaction are displayed immediately after each measurement, allowing amplification runs to be terminated or extended, as appropriate, during individual runs.
124Rotor-Gene from Corbett Research Dual Light source Excitation: 470 & 53032 x 0.2 ml plastic tubesAir heated, centrifugal mixingRotor-Gene from Corbett Research
125iCycler from BioRad Real Time Detection 1a. Excitation filters 1b. Emission filtersTungsten halogen light source( nm continuous)Microplate formatCycleriCycler from BioRad
126Biorad Instruments have recently launched an optical module that fits their standard thermal cycler and transforms it into a real-time RT-PCR system. This instrument is capable of generating and detecting a wider range of excitation frequencies than either the ABI 7700 or the Lightcycler. At present, it can monitor up to four different fluorescent reporters at any one time and can be used for any one of of the alternative fluorescent RT-PCR strategies. Furthermore, unlike the ABI 7700 which scans its 96 samples sequentially, this instrument can scan up to 96 samples simultaneously, with a sampling frequency that can be defined by the user.
127Mx4000™ Multiplex Quantitative PCR System from Stratagene Quartz tungsten halogen lamp (excitation range of 350 to 750nm)96 well platesFast cycling (90 min)CCD camera for capturing fluorescenceMx4000™ Multiplex Quantitative PCR System from Stratagene
128The ABI Prism 7700 (Perkin-Elmer–Applied Biosystems) contains a built-in thermal cycler with 96-well positions, and is able to detect fluorescence between 500 nm and 660 nm. Fluorescence is induced during the PCR by distributing laser light to all 96 samples contained in thin-walled reaction tubes via a multiplexed array of optical fibres. The resulting fluorescent emission returns via the fibres and is directed to a spectrograph with a charge-coupled device (CCD) camera. Because each well is irradiated sequentially, the dimensions of the CCD array can be used for spectral resolution of the fluorescent light. This instrument can be used for assays based on DNA-binding dyes, molecular beacons and hydrolysis probes.
132B. Oligonucleotide Hybridisation Probes I. Hydrolysis Probes (TaqMan)
133This strand is not shown below TaqMan ProbeRQForward PrimerReverse PrimerThis strand is not shown belowProbeThe TaqMan probe binds to ssDNA at a combined annealing and elongation step. It is degraded by the polymerase which releases the reporter dye (R) from the quencher (Q).
134Designing TaqMan Probes NewDesigning TaqMan ProbesTaqMan® Probe Design:Keep the G-C content in the 30—80% range.Avoid runs of an identical nucleotide especially GuanineDo not put Gs on the 5' end.Select the strand that gives the probe more Cs than Gs.For single-probe assays, Tm should be 68—70 °CPrimer Design:Choose the primers after designing the probe.Design the primers as close as possible to the probe without overlapping the probe.The Tm should be 58—60 °C.The five nucleotides at the 3' end should have no more than two Gand/or C bases.NOTE: Applied Biosystems provide Primer Express® for design of primers and probes in real time with Real Time Quantitative PCR systems (7700, 5700)
135B. Oligonucleotide Hybridisation Probes II. Strand Displacement Probes A. Roche Dual probe
136Microbe using identification16S rRNA genes Adjacent probes
137Designing Dual Adjacent Hybridisation Probes NewDesigning Dual Adjacent Hybridisation Probes(i) Identify useful Primer / probe regions (use any of the Primer software)(ii) Check primer / probe specificity using Fasta against Genbank database(iii) Check Tm using(iv) Check propenisity of probe to self anneal using Oligo Selection Program(v) Probe Tm’s should be near equal and 5-10C greater than primer Tm’s(vi) The 3’ end of the upstream probe should be labeled by fluorescein, which serves as the donor in the FRET and blocks extension from the probe(vii) the 5’ end of the downstream probe should be labeled by Cy5, which serves as the acceptor in the FRET, and the 3’ end of the probe should be phosphorylated to block extension the probes should be separated by one base(viii) the probes should be placed on one strand near a primer on the opposite strand.
139FITC probeCy5 probeForward PCR primerReverse PCR primerDesigning rRNA gene directed fluorroprobes for detection & identification of Campylobacter & Arcobacter by Real Time PCR
14021Figure 1: Continuous monitoring of fluorescence during PCR in which DNA templates from A. butzleri ATCC (--), C. jejuni ATCC (-+-) and A. skirrowii ATCC (-х-) show a significant increase in fluorescence emission whereas templates from E. coli (--), C. upsaliensis (--) and C. hyointestinalis (-o-) show only a marginal increase when compared to no DNA template control (-◊-) which shows no increase. Template DNA was prepared using the rapid boiling methodFigure 2. Derivative melting curves (-dF/dT) determined by the dissociation of fluorogenic adjacent hybridisation probes from the target amplicons enables discrimination of A. butzleri ATCC (Tm 68 oC), A. skirrowii (Tm 64 oC), C. jejuni ATCC and C. coli (Tm \ 66 oC) from each other. E. coli, C. upsaliensis, and C. hyointestinalis and template DNA produce no Tm.
142Forward PCR primerCy5 probeReverse PCR primerDesigning virulence gene directed fluorroprobes for detection, identification & differentiation of Campylobacter coli & Campylobacter jejuni from other species by Real Time PCR
14321Figure-1- Real time detection Real time detection of hippuricase gene for Campylobacter jejuni (-■-), Campylobacter coli (-▲-), Campyloacter hyointestinalis (-×-),Campylobacter upsaliansis(--),E. coli (-●-) and Negative control (-+-) (No templateFigure 2: Melting temperature of hippuricase gene for Campylobacter species
145B. Oligonucleotide Hybridisation Probes II. Strand Displacement Probes B. Hair Pin ProbesMolecular BeaconSunrise UniPrimerScorpionStem & LoopDuplexFRET Duplex
146The loop consists of target specific nucleotide (probe) sequences MOLECULAR BEACONSMolecular Beacons are hairpin structures composed of a nucleotide base paired stem and a target specific nucleotide loop.The loop consists of target specific nucleotide (probe) sequencesThe stem is formed by annealing of complementary nucleotide bases of the probe sequence. A fluorescent moiety (reporter)is attached to one end of the arm and a non-fluorescent quenching moiety is attached to the other arm. The stem keeps both the moieties in close proximity so that fluorescence is quenched(Loop)Stem
147Primer molecular beacon 5’3’Q5’3’3’5’RDenaturationPrimer molecular beaconannealingExtensionOperation of Molecular Beacon (MB): MB is non-fluorescent due to close proximity of the non-fluorescent quencer (Q) and the fluorescent Reporter. However when the probe denatures and the loop anneals to the target sequence of the amplicon, a conformational reorganization occurs separating the quencher from the fluorophore and thereby producing fluorescence which is proportional to the amplicons produced during PCR
148Q SUNRISE UNIPRIMER PROBE Similar to Molecular Beacon except that the stem contains a poly A (15 mer) tai. This tail is complimenatry to the polyT tail of one f the primers.QAAAAAAAAAAAAAAAPolyA Tail
149Sunrise UniPrimer Probe is a modification of Molecular Beacon Primer with polyT tailTTTTTTTTTTTTTTTSunrise Probe with polyA tail binds to the primer polyT tail at annealing.TTTTTTTTTTTTTTTAAAAAAAAAAAAAAQRhvQRAAAAAAAAAAAAAAThe Sunrise probe changes conformation during denaturation & quenching by DABCYL is removed allowing FITC to fluoresceSunrise UniPrimer Probe is a modification of Molecular Beacon
150Scorpion stem-loop format The template & probe denatureThe primer is part of the Scorpion probeScorpion stem-loop formatPrimer, stopper to prevent read PCR through, probe sequence, fluorophore & quencher (detection system).The primer is extendedThe primer bindsto the targetThe probe binds to the complimentary sequence of the DNA
151Duplex Scorpion Format Similar to the Ste-loop Scorpion except the probe sequence is part of the stem. There is no loop in this case.
152FRET Duplex Scorpions with 3 different versions of the quencher oligonucleotides
153Designing Stem Loop Molecular Beacon (MB) Probes New In general, design complexty: Dual adjacent > Taqman > Stem LoopIdeally, MBs should hybridise at their annealing temps (fluorescent) & free MBs should be closed (nonfluorescent)Use Oligo4.0 or “percent GC rule” to calculate that the loop sequence length (usually nucleotides) is such that it dissociates from its target at temperatures 7-10 oC higher then the annealing temp of the PCR.Add two complimentary arms on either side of the loop probe sequence. (usually 5-7 nucleotides; 5 GC rich stems melt between 55 & 60, 6 between 60 & 65 and 7 between 65 & 70). In order that it remains closed in the absence of the target, the length & GC content should be 7-10 oC higher then the annealing temp of the PCR. The melting temperature of the stem cannot be predicted by the “GC rule” as the stems forms by an intramolecular hybridisation eventThere should be no in between conformational changes, ie should always be the intended hairpin structure an d nothing in between.Commercial program available for making MB probes...\..\My Documents\My Pictures\BD100Tour.exe
158Advantages of Adjacent Probe Technique with Real Time PCR (Idaho -> Roche): 1. Rapid requiring < 30 mins in a Light Cycler2. rRNA and / or rRNA genes can be used = flexible3. Simultaneous detection, identification & quantitation4. PCR primer design + probe design = extremely specific assays possible5. Different flurodyes available. Multiplexing possible6. Population dynamics in an ecosystem can be followed7. Forms a powerful tool when used in conjunction with rRNA sequencing & FISH
160Pulsed Field Gel Electrophoresis (PFGE) agarose gel electrophoresis is a fundamental technique in molecular biology but is generally unable to resolve fragments greater than 20 kilobases in size (whole microbial genomes are usually greater than 1000 kilobases in size)PFGE (pulsed field gel electrophoresis) is a adaptation of conventional agarose gel electrophoresis that allows extremely large DNA fragments to be resolved (up to megabase size fragments)essential technique for estimating the sizes of whole genomes/chromosomes prior to sequencing and is necessary for preparing large DNA fragments for large insert DNA cloning and analysis of subsequent clonesalso a commonly used and extremely powerful tool for genotyping and epidemiology studies for pathogenic microorganisms
161Principle of PFGEtwo factors influence DNA migration rates through conventional gels- charge differences between DNA fragments- ‘molecular sieve’ effect of DNA poresDNA fragments normally travel through agarose pores as spherical coils, fragments greater than 20 kb in size form extended coils and therefore are not subjected to the molecular sieve effectthe charge effect is countered by the proportionally increased friction applied to the molecules and therefore fragments greater than 20 kb do not resolvePFGE works by periodically altering the electric field orientationthe large extended coil DNA fragments are forced to change orientation and size dependent separation is re-established because the time taken for the DNA to reorient is size dependent
163Principle of PFGEthe most important factor in PFGE resolution is switching time, longer switching times generally lead to increased size of DNA fragments which can be resolvedswitching times are optimised for the expected size of the DNA being run on the PFGE gelswitch time ramping increases the region of the gel in which DNA separation is linear with respect to sizea number of different apparatus have been developed in order to generate this switching in electric fields however most commonly used in modern laboratories are FIGE (Field Inversion Gel Electrophoresis) and CHEF (Contour-Clamped Homogenous Electrophoresis)
164CHEFSwitch TimeElectric Field 1Electric Field 2--------++++++++
165Preparation of DNA for PFGE ideally a genomic DNA preparation that contains a high proportion of completely or almost completely intact genome copies would be suitable for PFGEconventional means of DNA preparation are unsuitable for PFGE as mechanical shearing and low-level nuclease activity will result in fragmented DNA with an average size much smaller than an entire microbial genome (usually less than 200 kb in size)the solution to this is to prepare genomic DNA from whole cells in a semisolid matrix (ie. agarose) that eliminates mechanical shearinga very high concentration of EDTA is also used at all times in order to eliminate all nuclease activity
166Preparation of DNA for PFGE 1) intact cells are mixed with molten LMT agarose and set in a mold forming agarose ‘plugs’2) enzymes and detergents diffuse into the plugs and lyse cells3) proteinase K diffuses into plugs and digests proteins4) if necessary restriction digests are performed in plugs (extensive washing or PMSF treatment is required to remove proteinase K activity)5) plugs are loaded directly onto PFGE and run
167Preparation of DNA for PFGE for restriction digests, conventional enzymes are unsuitable as they cut frequently on an entire genome sequence producing DNA fragments that are far too small‘rare cutter’ restriction endonucleases cut genomic DNA with far less frequency than conventional restriction enzymes such as HindIII, BamHI etc.many rare cutter RE’s have 6-bp (or longer) recognition sites eg. NotI GCGGCCGCin many cases the frequency of cutting is highly species dependent eg. BamHI will cut far less frequently on a low GC% genome when compared to a intermediate or high GC content genomesuitable rare cutter enzymes therefore have to be determined experimentally for each new species being studied
170DNA Microarraya completely annotated microbial genome sequence, whilst a powerful scientific tool, still doesn’t provide all of the information needed to understand the complete biology of an organism as it essentially a static picture of the genomefor truly complete characterisation, the dynamic nature of gene expression within a microbial cell needs to be determinedmicroarray technology allows whole organism gene expression to be investigatedPCR products of every gene from a complete genome sequence are bound in a high density array on a glass slidethese arrays are probed with fluorescently labelled cDNA prepared from whole RNA under specific environmental conditionsthe level of cDNA for each ORF is then quantified using high resolution image scanners
171DNA MICROARRAYS TECHNIQUES: The development based on bioinformatics knowledge genes & genomes;High throughput & can analysise complex gene expression profilesThere are different formats for DNA high density microarrays:cDNA arrays (Stanford University development 1999): 0.5 – 5kbOligonucleotide synthesised (Genechip®) arrays (Affymetrix, 1998): baseOligomers / PNA, in situ or spottedAn example of how a microarray is made by in situ synthesis approach is shown as a movie. ..\..\My Documents\GeneChip.mov
172STEPS IN THE DNA ARRAY TECHNIQUE: Probe Selection - cDNA / oligo with known identify: Small oligos, cDNA, chromosomeChip Fabrication – Putting probes on the chip: photolithography, pipette, drop-touch, piezoelectric (inkjet), electricTarget – fluroscently labeled sample: RNA (mRNA) to cDNAAssay: Hybridisation, ligase, base addition, electric, electrophoresis, fluocytometry, PCR-DIRECT, TaqManReadout: FlurorescenceInformatics: Robotic controls, image processing, DBMA, WWW, bioinformaticsEXAMPLES:BioMerieux is developing for a water company a 4 h fecal indicator test using Affymetrix technology1cm2 has 400k oligonucleotide probes, small volumes of sample required limits the usefulnessTaqMan type: Leptospira for WHO regional reference laboratory, Campylobacter & Arcobacter for QHSS, Brisbane.
173An example of Microarray hybridisation a microarray containing 97% of the predicted ORF’s from Mycobacterium tuberculosis was used to investigate the response to the antituberculosis drug isoniazid (INH)INH was found to induce several genes related to outer lipid envelope biosynthesis – consistent with the drugs physiological mode of actiona number of additional genes were also induced which may provide potential drug targets in the future
174INH untreated - greenINH treated - redOverlayYellow = Red + Green (no change in expression)Green (untreated controls) ie expressed without INH treatmentRed = expressed as a result of INH treatmentThe effect of Isoniazid (INH) on the gene expression of Mycobacterium tuberculosis using DNA microarray technique
175The Future of DNA Microarrays New 1. Studies on the mechanism of toxicity of drugs to humans:INH is very safe, but like any medicine it can sometimes cause side effects. (yellowish skin, dark urine, vomiting, loss of appetite, nausea, changes in eyesight, unexplained fever, unexplained fatigue & stomach cramps). Human DNA arrays can be used to investigate the mechanism of toxicity on human which may not be possible using animal models.2. Studies on cyanobacterial toxins extracted from nature blooms:A human DNA array can be used for testing water which has been contaminated by cyanobacterial blooms before and after treatment. This will provide useful information on exposure levels (time & concentration).3. The role of “uncultured” viruses on different human cell lines:This will provide a rapid method for identifying the most susceptible cell lines that can be used to isolate the “offending” pathogen.
177What are cantilever arrays? Cantilever arrays are produced by microfabrication in silicon using dry- and wet-ething techniques. The cantilevers are 500 µm long, 100 µm wide and about 1 µm thick. The spring constant is 20 milliNewton per meter, resulting in a resonance frequency of about 4 kHz. The reproducibility of the resonance frequency from cantilever to cantilever within the array is better than 2%.Owing to their high flexibility, such cantilevers are appropriate for measuring tiny changes in surface stress. For such applications as mass determination, we have designed special cantilevers with a thickness of about 8 µm, producing a resonance frequency of around 50 kHz.
178Cantilevers are used for imaging in scanning force microscopy but is now being tested as a nanotech sensor. A thin flexible beam made of silicon coated with a sensor layer serves as a chemical sensor. Eight cantilevers aligned in a row form a nanomechanical cantilever sensor array, which can detect small amounts of analytes via very specific reactions. The analyte can also be characterized via its diffusion properties through the coating, e.g. a polymer layer.Nanomechanical cantilever array sensorIf analyte molecules dock on the surface of the cantilevers the surface stress at the interface changes, causing the cantilever to bend.The amount of bending is quantitated.
179DNA hybridisation cantilever array The binding of two complementary single stranded oligonucleotides can be observed in a setup of two cantilevers, each functionalized with a different synthetic oligonucleotide (red and blue). If the complementary oligonucleotide (green) is injected, it binds preferably to the red oligomer, but not to the blue one. This hybridization process involves bending of the cantilever due to steric and charging effects. If the complementary sequence to the blue strand is injected, the second cantilever bends.