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