75% off all plans

Molecular Diagnostic Methods

On this page

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.

Continue reading on OnCourse

Sign up for free to access the full lesson, plus unlimited questions, flashcards, AI-powered notes, and more.

CONTINUE READING — FREE

or get the app

Rezzy AI Tutor

Have doubts about this lesson?

Ask Rezzy, our AI tutor, to explain anything you didn't understand

Practice Questions: Molecular Diagnostic Methods

Test your understanding with these related questions

What is Northern blot used to detect?

1 of 5

Flashcards: Molecular Diagnostic Methods

1/10

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

browseSpaceflip

Enjoying this lesson?

Get full access to all lessons, practice questions, and more.

START FOR FREE
Rezzy AI Tutor
Molecular Diagnostic Methods – NEET-PG Microbiology Notes | Oncourse