Molecular Typing Methods

Intro to Molecular Typing - Germ Detective Kickoff

• Molecular Typing: Characterizing microbes at the molecular level (DNA, RNA, protein) to differentiate strains.
• Purpose:
  • Epidemiology: Track infection source & transmission.
  • Outbreak investigation & control.
  • Monitor antimicrobial resistance.
• Ideal Method Criteria: 📌 DR TIPES
  • Discriminatory power (distinguish unrelated strains)
  • Reproducibility (consistent results)
  • Typability (types most isolates)
  • Inexpensive & easy Interpretation
  • Portability (field use)
  • Ease of use (simple workflow)
  • Speed (rapid turnaround)

⭐ High discriminatory power means a method can distinguish between closely related but epidemiologically distinct strains.

Classic Genotyping - Old-School DNA Prints

These methods analyze DNA fragment patterns ("fingerprints") for microbial strain differentiation.

⭐ Pulsed-Field Gel Electrophoresis (PFGE) is often considered a gold standard for typing many bacterial pathogens due to its high discriminatory power, despite being labor-intensive.

PCR-Based Typing - Amplify & Identify

• RAPD (Random Amplified Polymorphic DNA)

  • Principle: Arbitrary primers amplify random DNA segments.
  • Pros/Cons: Simple, quick; low reproducibility.
  • Use: Basic strain differentiation.

• AFLP (Amplified Fragment Length Polymorphism)

  • Principle: Restriction digest of DNA, adapter ligation, then selective PCR of fragments.
  • Pros/Cons: Highly reproducible, high discrimination; technically complex.
  • Use: Detailed fingerprinting.

• rep-PCR (Repetitive Element PCR)

  • Principle: Primers target interspersed repetitive DNA sequences (e.g., BOX, ERIC, REP).
  • Pros/Cons: Simpler than AFLP; variable reproducibility.
  • Use: Bacterial strain typing, epidemiological studies.

• MLST (Multilocus Sequence Typing) 📌 MLST: 'Multiple Loci, Sure Typing'

  • Principle: Sequences internal fragments of ~7 housekeeping genes; allelic profiles (alleles at each locus) define Sequence Types (STs).
  • Pros/Cons: Unambiguous, portable data (sequence-based); higher cost, less resolution for very close strains.
  • Use: Definitive strain typing, global epidemiology, population genetics.

• spa-typing (S. aureus)

  • Principle: PCR amplification and sequencing of polymorphic X region of protein A gene (spa).
  • Pros/Cons: Rapid, reproducible, good for S. aureus/MRSA; species-specific.
  • Use: MRSA typing, local outbreak investigations.

⭐ Multilocus Sequence Typing (MLST) offers excellent inter-laboratory comparability and data portability due to its unambiguous sequence-based allelic profiles.

Sequencing-Based Typing - Genome Deep Dive

• 16S rRNA Gene Sequencing:
  • Principle: Compares variable regions of the 16S ribosomal RNA gene.
  • Applications: Bacterial identification, broad phylogenetic studies.
  • Limitations: Insufficient for fine discrimination of closely related strains.
• Whole Genome Sequencing (WGS): Offers ultimate resolution.
  • Principle: Complete DNA sequence of an organism's genome.
  • Analysis Types:
    • SNP analysis: Identifies single nucleotide polymorphisms.
    • cgMLST: Allelic profiling of conserved core genes.
    • wgMLST: Broader allelic profiling (core + accessory genes).
  • Advantages: Highest discriminatory power, identifies virulence/resistance genes, outbreak investigation.
  • Challenges: Complex bioinformatics, data storage, cost.

⭐ Whole Genome Sequencing (WGS) provides the highest possible resolution for molecular typing and can simultaneously identify virulence factors, resistance genes, and phylogenetic relationships.

High‑Yield Points - ⚡ Biggest Takeaways

• PFGE (Pulsed-Field Gel Electrophoresis) is a gold standard for bacterial outbreak typing, resolving large DNA fragments.
• RFLP (Restriction Fragment Length Polymorphism) differentiates strains by restriction enzyme digestion patterns of DNA.
• MLST (Multilocus Sequence Typing) offers high discrimination by sequencing multiple housekeeping genes for epidemiology.
• WGS (Whole Genome Sequencing) provides ultimate resolution in typing, crucial for complex outbreak investigations.
• Ribotyping analyzes rRNA gene variations, useful for bacterial species and strain typing.
• Spa typing is key for S. aureus, targeting protein A gene (spa) polymorphisms for strain delineation.