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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)
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
Phenotypic Methods
MorphologyGrowth CharacteristicsSerotypingPhage typingBacteriocin Biochemical Characteristicsphenol red carbohydrate, catalase production, oxidase
production, tests, methyl red test, nitrate reduction,starch hydrolysis, tryptophan hydrolysis, hydrogen sulfide production, citrate utilization, and litmus milk
Microbiological Analyses
Create microbe on plate
Streak on fresh plate
Gram StainBiologVitek
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
Microbiological Testing in Industry
How can Molecular Methods Improve Testing?
• Sterility testing –• Environmental testing –• Raw material testing –• Water testing –• Personnel monitoring –
When Genotyping Necessary?
Sterility Test positive……
Requires
Investigation, corrective and preventative action (best case)
Pharmaceutical Case Study
Investigation 1 Result - Biolog 16S Sequencing
Results Sterility test positive
Paenibacillus plyymxya 99%
Bacillus pumilus 99% (R) Bacillus pumilus 100% (F)
Tester isolate Bacillus subtilis 93%
Bacillus pumilus 99% (R) Bacillus pumilus 98%(F)
Environmental isolate
Paenibacillus macerans 97%
Bacillus circulans (R) 99% Bacillus circulans(F) 98%
Environmental isolate
Paenibacillus pabuli 100%
Paenibacillus pabuli (R) 98% Paenibacillus pabuli (F) 98%
Pharmaceutical Case Study Investigation 1
PFGE• The PFGE results
showed identical banding patterns by not1 digest and are the same strain of Bacillus pumilus
Pharmaceutical Case Study
Investigation 2Number 16S Sequencing ID
% Relatedness (Forward and Reverse)
Area Location Test BoxAIR PCR
1. M. luteus 99% F - 99% R Micro R400P Trays Different Strain
2. M. luteus 99% F - 100% R Micro R400P Trays Different Strain
3. M. luteus 99% F - 99% R Micro R400P Sterilins Different Strain
4. M. luteus 99% F - 100% R Micro R400P Drawers Different Strain
5. M. luteus 99% F – Reverse not tested
Micro R400P 30-35 Degree Inc
Different Strain
6. M. luteus 100% F - 99% R Micro R400P 30-35 Degree Inc
Different Strain
7. M. luteus 99% F – sequencing failure R
Micro R400P 30-35 Degree Inc
Different Strain
8. M. luteus 99% F - 100% R Micro R400P Test tube racks
Different Strain
9. M. luteus 99% F - 99% R Micro R400P Test tube racks
Different Strain
10. M. luteus 99% F - 100% R Micro R400P Wrists and Cheeks
Different Strain
11. M. luteus 99% F - 99% R Micro R404 Grey Floor Different Strain
12. M. luteus 99% F - 100% R Micro IPA Composite sprayer
Different Strain
17. M. luteus 99% F - 99% R Sterility Positive Day1
N/A N/A Identical
18. M. luteus 99% F - 99% R Sterility Positive Day 14
N/A N/A Identical
Pharmaceutical Case Study Investigation 2
BoxAIRM 1 2 3 4 6 7 8 9 10 11 12 13 14 16 17 18 M
PCR
Sequencing Method
Automatic Sequencing
Species Level ID
411 completed bacterial genomes
Sequencing rRNA Genes
Structural
Conserved regions
Universal
Not laterally transferred
How is Ribosomal DNA Sequencing Carried Out?
• Ribosomal 18S/16S or 28S/23SSequencing
0
3.6
2
Penicillium corylophilum.seqProbe 1.seqPenicillium hirsutum.seqPenicillium chrysogenum.seq
Pre-analysis by BlastN
Commercial Systems – Species
level IDSpecies Level IDsMicroseq – Applied Biosystems
Sherlock (DNA) – Midi Inc.
Riboprinter (strain level?) – DuPont Qualicon
Bax (food pathogens, 0157 etc. ) – DuPont Qualicon
Microseq and Sherlock-DNA
3 Databases Fungal – D2 region of large rRNA subunitBacterial – 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)
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 differentiationCommercial SystemRibotytping-RFLP principle
Sequencing to Strain Level Identification
• ITS (Internal transcribed spacer regions)
• between 18S and 28S
• Or 16S and 23S
RFLP
RFLP (Restriction Fragment Length Polymorphism)
Restriction digest of a) direct extracted DNAb) amplified selected genes via
PCR
Example of a PFGE Analysis on Staphylococcus aureus.
PFGE
Figure 2. Example of a real PFGE; drug
resistant Staphylococcus aureus. The molecular weight markers are digested
lambda phage (λ) and are given in kb. See the CDC web page with the original data .
BOXAIR-PCR
BOX-PCR (Amplification of BOX-cassettes)Rapid detection method with high reproducibility.
Commercial System - Strain level ID
Riboprinter
adheres to good manufacturing practice (CGMP) regulations (21 CFR part 11 for audit trails)
Ribotyping
•
Riboprinter Batch System Report
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