Clinical Application and Interpretationof Molecular Microbiological MethodsKurtD.Reed,M.DProfessorandViceChairmanDepartment of Pathology and LaboratoryMedicineUniversityofWisconsin-Madison,USA
Clinical Application and Interpretation of Molecular Microbiological Methods Kurt D. Reed, M.D. Professor and Vice Chairman Department of Pathology and Laboratory Medicine University of Wisconsin – Madison, USA
OutlineBrief history of the development of molecularmicrobiology Goals for molecular microbiology in the clinicallaboratoryMajor test platforms and methods Interpreting results -possibilities, practicalities andpitfallsWhat does the future hold?
Outline • Brief history of the development of molecular microbiology • Goals for molecular microbiology in the clinical laboratory • Major test platforms and methods • Interpreting results – possibilities, practicalities and pitfalls • What does the future hold?
Na 415s April 25, 1953NATURE737that the two lle sideby side with kentical z-co-ordinates, One ofthe pairmustbea purineand the otherapyrimidineforboeding tooccur.Thehydrogenbondtsaremade as fotlows:purine positionMOLECULARSTRUCTUREOFto pyrimidine position I; purine position 6 to pyrimidine positionNUCLEICACIDSIritisassumedthatthebasesontyoceurinthestructure nmeA Structure for Deoxyribose Nucleic Acidmostplausbtutmrimsthswthoathrtheenot configurations) it isfouend thatonlyspecific pairs ofbases E wish to suggest a structure for the salt or deoxyribocecan bond together.These pairs are: adenine (purine)with thymime/nucletcacld(D.N.A)Thisstructurehas novel features(pyrimidine),and guanine (purine) with cytosine (pyrimidine)which are ofconstderablebiological interest,In otherwordk ifanadenineforms one memberofapair,onAstructure for mucletc acid has already been pioposed byeither chain, tben on these assumptions the othermemher must bePauling and CoreyThey kindly made their manuseript availablethymine, simularly for gunnine nd cytosine.mhe sequenceoftous in advance ofpublication.Theirmodel consists or tareetnter-bases on a single chain, does not appear to beresticted in amytwined chains, with the phosphates near the nibre axis, and thewayHowever,ifoalyspecifiepainsofasescanbefomedtbases om the outsde, Inoueropinion this stnuctureis unsatistactoryrollowsthutthesequenceorbasesonmechalnisgivmmfoe two reasons:the srquenceon the other chaln is automatically dtermined(l) we bellevethat the material whidhiglves the X-raydiagrams bhasbeen foundexperimentally"that theratio orthe amoxeststhe salt, uot the tree acid.Without the ackdic hydrogen atoens it isofadenine to thymine,and theralioofguunine tocytosine,arenotcearutforswouldhodthesrucuretogeterespeciayatwuys very ckose to umity for deoxyribose nuckeic acid.as the negatively charged phosphates near the axis will repel eachB is probsbly mpossibleto build this structure with a riboseother. (2) Some of the van der Waals distancesappearto betoosugar in place orthe deoxyribose,as theextrs cxygen akom wouldamallmake too close avan der Waals contactAnotharthree-chainstructure has alsobeensuggested by FraserThe previously published X-ray dataon deoxyribose nuclei(in the press) In his model the phosphates are on the outside andacid are insufficient forarigorous test ofour structume.Sofar asthe bases on the insede, linked together by hydrogen bonds, Taiswe can tell, it ts roughly compatihle with the experimntad data.strecture as descibed is rather ill-definod, and for this reason webut ltmustbe reparded as eproved umntil has boen checkedshall not comment onitagainstmore exact resultxSome of these areglven ImtimeWeuish toput forand a raclikcaly different structurefor thefollowing, conmmications.We were not aware of the detaibs ofsalt or deoxyribose nucleic ackd.This structure has two helicalthe results presented there when we devised our structure,whichchains each coiled round the sameaxts (sae diagram) We huverests mainly though not emtinely oeputished experimeetal datamade the ususal chemical assumpiors.mamely,ttat eech chainand slereo-chemicalargumentsconsistsof phosphatediestergroups joining B-D-deoxyhusnotescaped ourmotice thatthespecific patrtng wehaveribofuranoseresidues wsth3'5linkagesTherwocules (butnxtpostudated immeliately suggest a posible copying mechanismtheir bases)are related by a dyad perendicular to the fitrdfor the genetie materiaiBoth chains follow rtghthianded helices, but owing to thedyad theFull detaits cf the stnucture, incladingthe conditions assumedsequencesftheatcemsinthewo dhainrun inoppostedirectioesin bullding it, togetherwith a setof.co-ordinates forthe atoms.Each chain loosely resenbles Furberg'swill be published elsenhertmodel No.1; that is, the bases are on theWe are much indeblod to Dr. Jemy Donctue for constantInside cr the helix and the phosphates onadvice and critikism especially on interatomic distances, We havethecutsideTheconfigurationathesugualso been stimuliated by a knowedge or the general mature of theand the atorns near it is close to Fusberg'sunpublished eperimentul resultsandKoss of Dx.M,HFstandard confieurationthe sugarbeingWilkins,Dr.R. .Frktin and their co-woekersatroughly perpendticular to the atacthiedKing's Colege, Londm,Ome ofus (a.D.w.) hus been aidedbyabase.There Is a residue on each chainfelowship frcmthe Natiomal Foundation foe Infantile Paralysisevery 3.4 A. in the z-dinection We have.D.WATSONassumed an angle of 36°between adjacayresidues in the same chain, so that theF.H,CCRICKstnucturerepeats after10residues on eachchain,that is,afler34AThedistanceofaMedical Research CouncifUrint for the Stucy othe Molecularphosphonus atom from the fibre axis is 10Sruxture of Biokgical Systems, Cavendish LaboraoryA. As the phosphates are on the ouside,Cambridge,April 2.cations have easy access tp them.>10,700citationsThe strudure is an open one,and txwater coutent is rather high,At lowerPalisg.LaalCere.LB.tm.11M6095tPos,UNAead SaJ8,water contenits we would expact the bases(tgs)to tilt so thse the structure could becoerugs,aaona,amore eompactCegtLfiedennorwe ZaeatS,thram G, anf Chaogut E.The novel feature ofthestructuze ts theBachin a epogsAes.9ste (a9tt)manner in which the two chairs are hekd*wyatCL.Ge.sL3s24952together by the purine and pyrimidineA.w..paech..Bbases, The planes of the bases are perpendicular io the fibre axis.'WRiM.FlRedExTicna.RakyAtaloo (os)They are joined together in pains.a single bse from one chainbeing bydropen-bonded to a single base fromn the other duin,scd中
> 10, 700 citations
Important Milestones in Molecular BiologyHost-controlled restriction-modification in bacteriophagesChemical and enzymatic DNA sequencingPolymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE),MLST andother genetic typing methodsRandom fragment sequencing and genome assembly“-omics technology (transcriptomes, proteomes,metabolomes)Next generation sequencing (NGS)Essentially every new discovery in molecularbiologyhas benefited the clinicallaboratory
Important Milestones in Molecular Biology • Host-controlled restriction-modification in bacteriophages • Chemical and enzymatic DNA sequencing • Polymerase chain reaction (PCR) • Pulsed-field gel electrophoresis (PFGE), MLST and other genetic typing methods • Random fragment sequencing and genome assembly • “-omics technology (transcriptomes, proteomes, metabolomes) • Next generation sequencing (NGS) • Essentially every new discovery in molecular biology has benefited the clinical laboratory
Goals for Molecular Microbiology in theClinical LaboratoryIdentifypathogens- Non-culturable,fastidiousandslow growing agents(HPV,Hepatitis B)- Highly infectious agents too dangerous to culture (BrucellaCoccidioides)Localizeinfectiousagentsintissue- e.g.Viruses,Toxoplasma.Quantify pathogens for prognostic and treatment purposes- HIV, CMV,Hepatitis B and CDifferentiate antigenically similar agents- HPV genotypes to determine cancer riskHospital and community epidemiologyAntiviral/antibacterialsusceptibilitytesting
Goals for Molecular Microbiology in the Clinical Laboratory • Identify pathogens – Non-culturable, fastidious and slow growing agents (HPV, Hepatitis B) – Highly infectious agents too dangerous to culture (Brucella, Coccidioides) • Localize infectious agents in tissue – e.g. Viruses, Toxoplasma, • Quantify pathogens for prognostic and treatment purposes – HIV, CMV, Hepatitis B and C • Differentiate antigenically similar agents – HPV genotypes to determine cancer risk • Hospital and community epidemiology • Antiviral/ antibacterial susceptibility testing
Practical Considerations for Patient Care. Reduce turn-around-times for results- Decrease length of stay- Reduce unnecessary antibiotic use and allow for morefocused treatment when it is necessaryImprove sensitivity and specificity- e.g. vastly improved detection of sexually transmittedinfectionsReducecosts-Moleculartests maybe expensive tothelaboratory butcan translate into cost savings to the institution: Standardize result reporting across hospitals- e.g. industry standards for quantification of viruses
Practical Considerations for Patient Care • Reduce turn-around-times for results – Decrease length of stay – Reduce unnecessary antibiotic use and allow for more focused treatment when it is necessary • Improve sensitivity and specificity – e.g. vastly improved detection of sexually transmitted infections • Reduce costs – Molecular tests may be expensive to the laboratory but can translate into cost savings to the institution • Standardize result reporting across hospitals – e.g. industry standards for quantification of viruses
Categories of Molecular Methods Hybridization methods - generally good foridentification, can be more sensitive than culturebut often not as sensitive compared to amplificationmethods. Early adoption of these methods by manyclinical labs.: Amplification methods - excellent sensitivity andspecificity. Contamination and workflow issues hadto be overcomebefore useful clinicallySequencing and enzymatic digestion of nucleicacids -fueling an explosion of knowledge inpathogen discovery, mechanisms of disease andmolecular epidemiology. Current use by largelaboratories and reference labs
Categories of Molecular Methods • Hybridization methods – generally good for identification, can be more sensitive than culture, but often not as sensitive compared to amplification methods. Early adoption of these methods by many clinical labs. • Amplification methods – excellent sensitivity and specificity. Contamination and workflow issues had to be overcome before useful clinically. • Sequencing and enzymatic digestion of nucleic acids – fueling an explosion of knowledge in pathogen discovery, mechanisms of disease and molecular epidemiology. Current use by large laboratories and reference labs
NucleicAcid HybrizationOrganismADNAOrganismBDNA刷品盛图暖政Heat to separate strands.Combinesinglestrands of DNACool to allow renaturationofdouble-stranded DNA.Determine degreeofhybridization.Completehybridization:Partial hybridization:No hybridization:organismsidenticalorganismsrelatedorganismsunrelatedLookssimplebutmanythingscangowrong.Needhighlyaccurateandconsistentresultstobeusefulintheclinicalsetting
Nucleic Acid Hybrization Looks simple but many things can go wrong. Need highly accurate and consistent results to be useful in the clinical setting
Steps Involved in Hybridization Reactions1)ProduceandlabelsingleHybridizationstrandedprobesrRNA2)Preparesinglestranded60°℃CHybridstarget nucleic acid1hourHybridizeDNA3)Annealtargetan probeYXProbeunderappropriateconditionsof stringencyHVBRIDIZED PROBESelec?DelesX4) Detect hybridizationLighireactionUNHYBRIDIZED PROBECaAoSelseBeteata) Solution formatb)SolidsupportformatSolutionformat hybridization
Steps Involved in Hybridization Reactions 1) Produce and label single stranded probes 2) Prepare single stranded target nucleic acid 3) Anneal target an probe under appropriate conditions of stringency 4) Detect hybridization reaction a) Solution format b) Solid support format Solution format hybridization
Southern Blot Hybridization"Blot"DNAfragments fromToomanysteps,tooagarosegelontomembranetimeconsuming,andPlace DNA fragmentsonanagarosegel andtoo subjectiveto beseparatebyelectrophoresispracticalin manylaboratories.MembraneimprintedwithDNAbandsDetection (the methoddepends onthetype ofprobeyou use)revealsabandwhereyour probebound to thetargetsequence.Addalabeledprobetothemembrane (in buffersolution)
Southern Blot Hybridization Too many steps, too time consuming, and too subjective to be practical in many laboratories