Molecular Identification Methods

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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)

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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

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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

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Microbiological Analyses
Create microbe on plate
Streak on fresh plate
Biolog
Vitek
Gram Stain
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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

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Microbiological Testing in IndustryHow can
Molecular Methods Improve Testing?

• Sterility testing
• Environmental testing
• Raw material testing
• Water testing
• Personnel monitoring

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When Genotyping Necessary?

• Sterility Test positive
• Requires
• Investigation, corrective and preventative action
  (best case)

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Pharmaceutical Case Study Investigation 1
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Pharmaceutical Case Study Investigation 1 PFGE

• The PFGE results showed identical banding
  patterns by not1 digest and are the same strain
  of Bacillus pumilus

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Pharmaceutical Case Study Investigation 2
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Pharmaceutical Case Study Investigation 2 BoxAIR
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PCR
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Sequencing Method
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Automatic Sequencing
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Species Level ID
Sequencing rRNA Genes
411 completed bacterial genomes
Structural Conserved regions Universal Not
laterally transferred
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How is Ribosomal DNA Sequencing Carried Out?

• Ribosomal 18S/16S or 28S/23S

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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

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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)

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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

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Sequencing to Strain Level Identification

• ITS (Internal transcribed spacer regions)
• between 18S and 28S
• Or 16S and 23S

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RFLP

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

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Example of a PFGE Analysis on Staphylococcus
aureus.
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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 .  
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BOXAIR-PCR

• BOX-PCR (Amplification of BOX-cassettes)
• Rapid detection method with high reproducibility.

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

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Riboprinter Batch System Report
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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