Question 11: Two different specimens from two patients are received. Which of the following techniques can confirm they are from different individuals (in the context of Mycobacterium tuberculosis)?
- A. RFLP (Correct Answer)
- B. Pyrosequencing
- C. Mutation analysis
- D. RAPD
Explanation: ***Correct: RFLP***
- **RFLP** (Restriction Fragment Length Polymorphism), particularly using the **IS6110 insertion sequence**, is the traditional gold standard tool for **epidemiological strain typing** of *M. tuberculosis*.
- Confirmation that two specimens are from different individuals relies on visualizing two distinct banding patterns, indicating different arrangements of the **IS6110** elements in their respective genomes.
- RFLP has **high discriminatory power** for differentiating between strains and establishing epidemiological links.
*Incorrect: Mutation analysis*
- This technique primarily focuses on detecting **point mutations** in specific genes (e.g., *rpoB*, *katG*) to determine drug susceptibility or resistance patterns.
- While differences in resistance profiles suggest different strains, it lacks the **high discriminatory power** of RFLP or VNTR for broad epidemiological linkage assessment.
*Incorrect: Pyrosequencing*
- Pyrosequencing is a rapid method of DNA sequencing often used to detect **single nucleotide polymorphisms (SNPs)** or specific mutations, such as those related to drug resistance.
- It is utilized for sequencing short regions and is not the primary technique for comparing the **large-scale genomic structures** necessary for definitive strain fingerprinting.
*Incorrect: RAPD*
- **RAPD** (Random Amplification of Polymorphic DNA) is an older PCR-based method that uses arbitrary primers to generate a pattern of amplified DNA fragments.
- While used for strain comparison in some bacteria, RAPD is generally considered **less reproducible** and has **lower discriminatory power** than RFLP or VNTR for *M. tuberculosis* typing.