In situ Hybridization: Allows pathogens to be identified and localizedwithin tissues.IdentificationofbacteriafromLocalizationofinvasiveE.coliincolonictissuepositivebloodcultures PNA-FiSH
In situ Hybridization • Allows pathogens to be identified and localized within tissues. Identification of bacteria from positive blood cultures PNA-FISH Localization of invasive E. coli in colonic tissue
Applications of Hybridization Techniques Direct detection of pathogens: e.g. Group AStreptococcus, N. gonorrhea, C. trachomatis.Replaced traditional culture in many labs. ldentification of culture isolates - dimorphicfungi, mycobacteria: Advantages included rapid turn-around-time forresults. Good sensitivity and specificitycompared to culture.: Disadvantages include relative insensitivitycompared to amplification techniques
Applications of Hybridization Techniques • Direct detection of pathogens: e.g. Group A Streptococcus, N. gonorrhea, C. trachomatis. Replaced traditional culture in many labs. • Identification of culture isolates – dimorphic fungi, mycobacteria • Advantages included rapid turn-around-time for results. Good sensitivity and specificity compared to culture. • Disadvantages include relative insensitivity compared to amplification techniques
Amplification Techniques.Target amplification- PCR - thermal cycling required. (lnitial fears ofunacceptableratesof contaminationhavebeenovercomeby a combination of chemical/enzymatic decontaminationof amplicons,single tube methods and workflow design.)-Isothermalamplification.Nucleicacidsequencebasedamplification(NASBA).Transcription mediated amplification (TMA): Strand displacement amplification (SDA).Loopmediated isothermalamplification (LAMP)Signal amplificationProbeamplification
Amplification Techniques • Target amplification – PCR – thermal cycling required. (Initial fears of unacceptable rates of contamination have been overcome by a combination of chemical/enzymatic decontamination of amplicons, single tube methods and workflow design.) – Isothermal amplification • Nucleic acid sequence based amplification (NASBA) • Transcription mediated amplification (TMA) • Strand displacement amplification (SDA) • Loop mediated isothermal amplification (LAMP) • Signal amplification • Probe amplification
16S rRNA GenesFoundin allbacteria· Accumulate mutations SLOWLY hence theyhave been used as “"molecular clocks" Conserved regions of the gene are targets of"broad-range" primers for any/all bacteria Highly variable regions of the gene provideunique signature sequences to identify thebacterium
16S rRNA Genes • Found in all bacteria • Accumulate mutations SLOWLY hence they have been used as “molecular clocks” • Conserved regions of the gene are targets of “broad-range” primers for any/all bacteria • Highly variable regions of the gene provide unique signature sequences to identify the bacterium
Bacterial genomeK16SrRNAgenefoundin>rRNAmultiplecopiesonthegenomeUnknownbacterium~1500bpgenecontainingconservedandvariableregionsConserved =target of PCR primers Variable =unique'signature'or ID