Molecular Diagnostic Methods

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Molecular Diagnostic Methods - PCR Powerhouse

  • Polymerase Chain Reaction (PCR): In vitro exponential amplification of specific DNA/RNA.
    • Core Principle: Enzymatic replication of a target nucleic acid sequence.
    • Key Components: Template (DNA/cDNA), primers, Taq polymerase (heat-stable), dNTPs, Mg²⁺.
  • PCR Cycle: Repeated 25-35 times. Results in $2^n$ fold amplification (n=cycles).
  • Variants & Uses:
    • RT-PCR (Reverse Transcriptase PCR): RNA detection (converts RNA to cDNA first). E.g., HIV, Influenza.
    • qPCR (Real-Time PCR): Quantifies DNA/RNA during amplification using fluorescence.
    • Applications: Rapid pathogen ID (TB, Chlamydia), genetic testing, oncology. PCR process showing DNA amplification cycles

⭐ Taq polymerase, isolated from Thermus aquaticus, is thermostable, withstanding high denaturation temperatures, making PCR automation possible.

Molecular Diagnostic Methods - Target Finders

  • Nucleic Acid Probes: Labelled (e.g., $P^{32}$, biotin, fluorophores) ssDNA/RNA fragments; bind specific complementary target sequences.
  • Hybridization: Process of probe annealing to target nucleic acid.
    • Solid-phase: Target immobilized on a membrane.
    • In-situ: Target within intact cells or tissues.
  • Blotting Techniques: For detecting specific DNA/RNA sequences.
    • Southern Blot: DNA detection.
-   **Northern Blot:** RNA detection (no restriction digestion needed).
-   **Dot/Slot Blot:** Rapid detection without size separation.
> ⭐ 📌 SNoW DRoP Mnemonic: **S**outhern Blot = **D**NA; **N**orthern Blot = **R**NA; **W**estern Blot = **P**rotein.
  • In Situ Hybridization (ISH): Localizes nucleic acids within cells/tissues.
    • FISH (Fluorescent ISH): Uses fluorescent probes. Applications: Gene mapping, detecting aneuploidies (e.g., Trisomy 21), pathogen identification. FISH direct and indirect labeling methods diagram technique showing probes targeting specific DNA sequences in chromosomes)
  • Microarrays (Gene Chips): Miniaturized arrays with thousands of probes; for high-throughput gene expression analysis, SNP genotyping.

Molecular Diagnostic Methods - Code Crackers

  • Principle: "Crack the code" of microbial nucleic acids (DNA/RNA).
  • Uses: ID, resistance, virulence, typing, epidemiology.

Microorganism Identification Methods

  • 1. Hybridization (Known Sequences):
    • Probes detect specific DNA/RNA.
    • Techniques: Southern/Northern blots, FISH, DNA microarrays.
  • 2. Amplification (Signal Boost):
    • PCR: Exponential DNA/RNA increase. Key types: RT-PCR (RNA), qPCR (quantifies).
    • Alternatives: LCR, TMA, NASBA.
  • 3. Sequencing (Unknown Sequences):
    • Sanger: Dideoxy chain termination. Gold standard for single genes.
    • NGS: High-throughput, for genomes/metagenomes.
  • 4. Genotyping/Typing (Strain Variation):
    • RFLP, PFGE: Restriction enzyme patterns.
    • MLST: Allelic variation in housekeeping genes. 📌 "Many Loci Sequenced Together".

    ⭐ PFGE is considered the gold standard for epidemiological typing of many bacterial pathogens.

Molecular Diagnostic Methods - Pathogen Hunt

Rapidly identifies pathogens by detecting their unique DNA/RNA, crucial for timely and targeted therapy.

  • Core Principle: Detect pathogen-specific nucleic acids.
  • Key Methods:
    • PCR (Polymerase Chain Reaction): Highly sensitive & specific.
      • RT-PCR: For RNA viruses (e.g., HIV, Influenza, SARS-CoV-2).
      • qPCR (Real-Time PCR): Quantifies pathogen load (viral load monitoring).
    • Sequencing (NGS, Sanger): Definitive ID, tracks outbreaks, detects resistance mutations.
    • LAMP (Loop-mediated Isothermal Amplification): Rapid, point-of-care testing.
  • Advantages: Speed, detects non-culturable/fastidious organisms, high throughput.
  • Applications: Diagnosis of sepsis, meningitis, TB, viral infections; antimicrobial resistance profiling.

⭐ PCR can detect as few as 1-10 copies of microbial DNA/RNA per sample, vital for early diagnosis even before seroconversion or in paucibacillary disease.

Molecular Diagnostic Methods Workflow

High‑Yield Points - ⚡ Biggest Takeaways

  • PCR: Core for amplifying DNA/RNA; offers high sensitivity in detecting pathogens.
  • Real-time PCR (qPCR): Allows quantification of microbial load, crucial for monitoring treatment response.
  • Multiplex PCR: Detects multiple pathogens simultaneously, useful for syndromic panels.
  • Nucleic Acid Hybridization Probes (e.g., FISH): Identify specific microbial sequences directly in samples.
  • Gene Sequencing (Sanger/NGS): Essential for genotyping, resistance mutation detection, and epidemiological tracing.
  • Molecular Typing (PFGE, MLST, Ribotyping): Crucial for strain differentiation during outbreak investigations.

Practice Questions: Molecular Diagnostic Methods

Test your understanding with these related questions

What is Northern blot used to detect?

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Flashcards: Molecular Diagnostic Methods

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If the second sputum sample for CB NAAT is Rif indeterminate/inconclusive, next step is:_____

TAP TO REVEAL ANSWER

If the second sputum sample for CB NAAT is Rif indeterminate/inconclusive, next step is:_____

do Line probe assay or liquid culture

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