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Title:

Molecular Identification Methods

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... Strain Level IDs Sequencing ITS region - Strain level identification RFLP Restriction Fragment Length Polymorphism Identification and differentiation PFGE ... – PowerPoint PPT presentation

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Title: Molecular Identification Methods


1
(No Transcript)
2
Molecular Identification Methods
  • Confirmation of identity for commonly used
    laboratory strains should ideally be done at the
    level of genotypic analysis... Pharmacopeial
    Forum, Volume 30 (5) 2004 Microbiological Best
    Laboratory Practices, Page 1717)

3
Overview
  • Current phenotypic methods/ Problems
  • Molecular case studies in industry
  • PCR/Sequencing techniques
  • Species level ID and Commercial systems
  • Strain level ID and Commercial system

4
Phenotypic Methods
  • Morphology
  • Growth Characteristics
  • Serotyping
  • Phage typing
  • Bacteriocin
  • Biochemical Characteristics
  • phenol red carbohydrate, catalase production,
    oxidase production, tests, methyl red test,
    nitrate reduction,starch hydrolysis, tryptophan
    hydrolysis, hydrogen sulfide production, citrate
    utilization, and litmus milk

5
Microbiological Analyses
Create microbe on plate
Streak on fresh plate
Biolog
Vitek
Gram Stain
6
Problems With Microbiological Testing in QA
Laboratories
  • Gram staining, messy, time consuming
  • Biolog/Vitek time consuming 2-3 days
  • Large amount of isolates unidentifiable
  • Only identifiable to genus, species level
  • Some non-specific, moulds take 2-3 weeks, many
    repeats

7
Microbiological Testing in IndustryHow can
Molecular Methods Improve Testing?
  • Sterility testing
  • Environmental testing
  • Raw material testing
  • Water testing
  • Personnel monitoring

8
When Genotyping Necessary?
  • Sterility Test positive
  • Requires
  • Investigation, corrective and preventative action
    (best case)

9
Pharmaceutical Case Study Investigation 1
10
Pharmaceutical Case Study Investigation 1 PFGE
  • The PFGE results showed identical banding
    patterns by not1 digest and are the same strain
    of Bacillus pumilus

11
Pharmaceutical Case Study Investigation 2
12
Pharmaceutical Case Study Investigation 2 BoxAIR
13
PCR
14
Sequencing Method
15
Automatic Sequencing
16
Species Level ID
Sequencing rRNA Genes
411 completed bacterial genomes
Structural Conserved regions Universal Not
laterally transferred
17
How is Ribosomal DNA Sequencing Carried Out?
  • Ribosomal 18S/16S or 28S/23S

18
Commercial Systems Species level ID
  • Species Level IDs
  • Microseq Applied Biosystems
  • Sherlock (DNA) Midi Inc.
  • Riboprinter (strain level?) DuPont Qualicon
  • Bax (food pathogens, 0157 etc. ) DuPont
    Qualicon

19
Microseq and Sherlock-DNA
  • 3 Databases
  • Fungal D2 region of large rRNA subunit
  • Bacterial 16S full sequence (1500bp), 16S
    partial sequence (500bp)
  • Microseq Software and Database adheres to good
    manufacturing practice (CGMP) regulations (21 CFR
    part 11 for audit trails)

20
Strain Level IDs
  • Sequencing ITS region
  • - Strain level identification
  • RFLP Restriction Fragment Length Polymorphism
  • Identification and differentiation
  • PFGE Pulse Field Gel Electrophoresis
  • Epidemiological studies
  • BoxAIR-PCR BoxA Inverse Repeat PCR
  • Identification and differentiation
  • Commercial System
  • Ribotytping
  • -RFLP principle

21
Sequencing to Strain Level Identification
  • ITS (Internal transcribed spacer regions)
  • between 18S and 28S
  • Or 16S and 23S

22
RFLP
  • RFLP (Restriction Fragment Length Polymorphism)
  • Restriction digest of
  • direct extracted DNA
  • amplified selected genes via PCR

23
Example of a PFGE Analysis on Staphylococcus
aureus.
24
PFGE
                                                
                                                  
                                                  
          

                                                
                                             Figur
e 2. Example of a real PFGE drug resistant
Staphylococcus aureus. The molecular weight
markers are digested lambda phage (?) and are
given in kb. See the CDC web page with the
original data .  
25
BOXAIR-PCR
  • BOX-PCR (Amplification of BOX-cassettes)
  • Rapid detection method with high reproducibility.

26
Commercial System - Strain level ID
adheres to good manufacturing practice (CGMP)
regulations (21 CFR part 11 for audit trails)
Riboprinter
27
Ribotyping

28
Riboprinter Batch System Report
29
Summary
  • Species Level ID
  • Sequencing 16S/5S/23S genes (bacteria)
  • Sequencing 18S/5.8S/28S (fungal)
  • Strain Level ID
  • Sequencing ITS region
  • PFGE Pulse Field Gel Electrophoresis
  • RFLP Restriction Fragment Length Polymorphism
  • BoxAIR-PCR BoxA Inverse Repeat PCR
  • Ribotytping
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