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Paper I — 2024 June (Supplementary) (2019 Scheme) — Q5

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Paper I
2024 June (Supplementary) (2019 Scheme) · 100 marks · 180 min

Question

List molecular methods used in laboratory diagnosis of infections. Briefly mention principle of each method. Discuss advantages and disadvantages of each method. (1+3+2+2)

Q58 marksShort Essays

Answer

Molecular methods used in laboratory diagnosis of infections

  1. Polymerase Chain Reaction (PCR) Principle: in-vitro enzymatic amplification of a specific target DNA sequence using sequence-specific primers, a heat-stable DNA polymerase (Taq polymerase), and repeated cycles of denaturation, annealing, and extension, producing millions of copies of the target sequence for detection. Advantages: extremely high sensitivity and specificity; rapid results; can detect organisms that are difficult/impossible to culture; detects very low pathogen loads. Disadvantages: requires expensive equipment/reagents and trained personnel; risk of contamination causing false positives; does not distinguish viable from non-viable organisms.

  2. Real-time PCR (qPCR) Principle: PCR combined with fluorescent probe/dye detection, allowing real-time quantification of amplified product as the reaction proceeds, based on the cycle threshold (Ct) value. Advantages: quantitative (viral load estimation), faster (no post-amplification gel electrophoresis needed), reduced contamination risk (closed-tube system). Disadvantages: higher equipment cost than conventional PCR; requires careful probe/primer design.

  3. Nucleic Acid Hybridization (e.g., DNA probes, in-situ hybridization) Principle: a labelled single-stranded nucleic acid probe binds (hybridizes) specifically to a complementary target sequence in the sample, and the label (radioactive, fluorescent, or enzymatic) is then detected. Advantages: relatively simple, can be applied directly to clinical/tissue specimens (in-situ hybridization localizes the organism within tissue). Disadvantages: lower sensitivity than PCR-based amplification methods (no amplification step); labour-intensive for some formats.

  4. Line Probe Assay (LPA) / Gene sequencing Principle: amplified product is hybridized to multiple immobilized probes on a strip (LPA) to detect specific mutations (e.g., drug-resistance mutations in M. tuberculosis), or the amplified DNA is directly sequenced to determine its exact base sequence. Advantages: provides detailed genotypic information, including drug-resistance profiling; sequencing gives definitive identification/strain typing. Disadvantages: relatively expensive and technically demanding; sequencing requires specialized bioinformatics support for interpretation.

(Other valid examples: nucleic acid sequence-based amplification (NASBA); loop-mediated isothermal amplification (LAMP) — rapid, does not require thermal cycling equipment, useful in resource-limited settings but can have lower specificity if not carefully optimized.)

  1. 1General Properties of Viruses and Virus Host Interaction

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