Tuberculosis is a bacterial infection that is caused by Mycobacterium tuberculosis which can attack the kidney, liver, etc., but when it infects the lungs, it causes tuberculosis. Tuberculosis is an infectious disease that can spread easily when people gather in crowds or where people live in crowded conditions. Many molecular markers can identify or differentiate the different types of bacterial strains that affect the person in different regions or the same region. The main aim of this study is to assess the applicability of IS6110-based RFLP analysis to sensitive strains of M. tuberculosis in different regions of India. DNA fingerprinting form of mycobacterium tuberculosis is a powerful epidemiologic tool used to detect and control tuberculosis. For this study, 38 samples from different age groups and sexes were collected and studied. These samples were processed, stained, cultured, DNA isolated, and amplified by PCR. These DNAs were obtained by targeting the insertion sequence IS6110. From 38 samples, 36 were PCR positive, whereas 2 were IS6110 PCR negative. 8 out of 9 untreated cases showed PCR positivity compared to smear and culture positivity. The DNA fingerprinting was used to assess epidemiologically linked case pairs of tuberculosis. The matched DNA fingerprints of the source and secondary confirm the transmission. RFLP analysis using the repetitive DNA element IS6110 in M. tuberculosis has been a powerful tool for confirming the result of standard epidemiological investigation. This standardized technique generates strain-specific patterns by creating variability in genomic position and number of IS6110. This technique compares results and identifies specific strains with unusual properties like high infectivity, virulence, or drug resistance.

1. Ryu YJ. Diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms. Tuberculosis and respiratory diseases. 2015 Apr;78(2):64.
2. Oommen S, Banaji N. Laboratory diagnosis of tuberculosis: advances in technology and drug susceptibility testing. Indian journal of medical microbiology. 2017 Jul 1;35(3):323-31.
3. Shah SZ, Jabbar B, Ahmed N, Rehman A, Nasir H, Nadeem S, Jabbar I, Rahman ZU, Azam S. Epidemiology, pathogenesis, and control of a tick-borne disease-Kyasanur Forest disease: current status and future directions. Frontiers in cellular and infection microbiology. 2018 May 9;8:149.
4. Aravena P, Pulgar R, Ortiz-Severín J, Maza F, Gaete A, Martínez S, Serón E, González M, Cambiazo V. PCR-RFLP detection and genogroup identification of Piscirickettsia salmonis in field samples. Pathogens. 2020 May 8;9(5):358.
5. Jagielski T, Van Ingen J, Rastogi N, Dziadek J, Mazur PK, Bielecki J. Current methods in Mycobacterium tuberculosis and other mycobacteria molecular typing. BioMed research is international. 2014 Oct;2014.
6. Bahmani N, Mirnejad R, Arabestani MR, Mohajerie P, Hashemi SH, Karami M, Alikhani MY. Comparison of PCR-RFLP and PFGE for determining the clonality of Brucella isolates from human and livestock specimens. Saudi Journal of Biological Sciences. 2019 Feb 1;26(2):256-62.
7. Sharma-Kuinkel BK, Rude TH, Fowler VG. Pulse field gel electrophoresis. The Genetic Manipulation of Staphylococci: Methods and Protocols. 2016:117-30.
8. Byrne AS, Goudreau A, Bissonnette N, Shamputa IC, Tahlan K. Methods for detecting mycobacterial mixed strain infections–a systematic review. Frontiers in genetics. 2020 Dec 21;11:600692.
9. Ei PW, Aung WW, Lee JS, Choi GE, Chang CL. Molecular strain typing of Mycobacterium tuberculosis: a review of frequently used methods. Journal of Korean Medical Science. 2016 Nov;31(11):1673.
10. Jagielski T, Van Ingen J, Rastogi N, Dziadek J, Mazur PK, Bielecki J. Current methods in Mycobacterium tuberculosis and other mycobacteria molecular typing. BioMed research is international. 2014 Oct;2014.
11. Pingoud A, Wilson GG, Wende W. Type II restriction endonucleases—a historical perspective and more. Nucleic acids research. 2014 Jun 26;42(12):7489-527.
12. Roychowdhury T, Mandal S, Bhattacharya A. Analysis of IS 6110 insertion sites provides a glimpse into Mycobacterium tuberculosis's genome evolution. Scientific reports. 2015 Jul 28;5(1):12567.
13. Kralik P, Ricchi M. A basic guide to real-time PCR in microbial diagnostics: definitions, parameters, and everything. Frontiers in microbiology. 2017 Feb 2;8:239909.
14. Chauhan A, Chauhan DS, Parashar D, Gupta P, Sharma VD, Sachan AS, Gupta R, Agarawal BM, Katoch VM. DNA fingerprinting of Mycobacterium tuberculosis isolates from Agra region by is 6110 probe. Indian Journal of Medical Microbiology. 2004 Oct 1;22(4):238-40.
15. Vestal AL. Procedures for the isolation and identification of mycobacteria. Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, Bureau of Laboratories, Training and Consultation Division; 1977.
16. Van Soolingen D, Hoogenboezem T, De Haas PE, Hermans PW, Koedam MA, Teppema KS, Brennan PJ, Besra GS, Portaels F, Top J, Schouls LM. A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa. International Journal of Systematic and Evolutionary Microbiology. 1997 Oct;47(4):1236-45.
17. Wang HyeYoung WH, Uh Young UY, Kim SeoYong KS, Shim TaeSun ST, Lee HyeYoung LH. Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria.
18. Minnikin DE. Good fellow M. Lipid composition in the classification of acid-fast bacteria. Good fellow M, Board RG.
19. Eddabra R, Ait Benhassou H. Rapid molecular assays for detection of tuberculosis. Pneumonia. 2018 May 25;10(1):4.
20. Kabir S, Uddin MK, Chisti MJ, Fannana T, Haque ME, Uddin MR, Banu S, Ahmed T. Role of PCR method using IS6110 primer in detecting Mycobacterium tuberculosis among the clinically diagnosed childhood tuberculosis patients at an urban hospital in Dhaka, Bangladesh. International Journal of Infectious Diseases. 2018 Mar 1;68:108-14.
21. Ei PW, Aung WW, Lee JS, Choi GE, Chang CL. Molecular strain typing of Mycobacterium tuberculosis: a review of frequently used methods. Journal of Korean medical science. 2016 Nov;31(11):1673.
22. Das S, Paramasivan CN, Lowrie DB, Prabhakar R, Narayanan PR. IS6110 restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, south India. Tubercle and Lung disease. 1995 Dec 1;76(6):550-4.
23. Katoch VM, Singh D, Chauhan DS, Sharma VD, Singh HB, Das R, Srivastava K. Newer DNA fingerprinting techniques for tuberculosis-relevance in control. Multi-drug resistance in emerging and re-emerging diseases. 2000:87-96.
24. Supply P, Magdalena J, Himpens S, Locht C. Identification of novel intergenic repetitive units in a mycobacterial two?component system operon. Molecular microbiology. 1997 Dec;26(5):991-1003.
25. Good fellow M, Lawrence G, Wayae. Taxonomy and nomenclature in biology of mycobacteria, C. Ratledge & J. Stanford. Academic presss London. 1882. 1:417-521.
26. Sola C, Filliol I, Legrand E, Mokrousov I, Rastogi N. Mycobacterium tuberculosis phylogeny reconstruction based on combined numerical analysis with IS 1081, IS 6110, VNTR, and DR-based spoligotyping suggests the existence of two new phylogeographical clades. Journal of molecular evolution. 2001 Dec;53:680-9.
27. Chevrel-Dellagi D, Abderrahman AM, Haltiti RA, Koubaji HI, Gicquel B, Dellagi K. Large-scale DNA fingerprinting of Mycobacterium tuberculosis strains as a tool for epidemiological studies of tuberculosis. Journal of clinical microbiology. 1993 Sep;31(9):2446-50.
28. Small PM, Hopewell PC, Singh SP, Paz A, Parsonnet J, Ruston DC, Schecter GF, Daley CL, Schoolnik GK. The epidemiology of tuberculosis in San Francisco--a population-based study using conventional and molecular methods. New England Journal of Medicine. 1994 Jun 16;330(24):1703-9.
29. Yang ZH, Mtoni I, Chonde M, Mwasekaga M, Fuursted K, Askgård DS, Bennedsen J, De Haas PE, Van Soolingen D, Van Embden JD. DNA fingerprinting and phenotyping of Mycobacterium tuberculosis isolates from human immunodeficiency virus (HIV)-seropositive and HIV-seronegative patients in Tanzania. Journal of Clinical Microbiology. 1995 May;33(5):1064-9.
30. Krishnan MY, Radhakrishnan I, Joseph BV, Gk ML, Kumar R A, Mundayoor S. Combined use of Amplified Fragment Length Polymorphism and IS 6110-RFLP in fingerprinting clinical isolates of Mycobacterium tuberculosis from Kerala, South India. BMC Infectious Diseases. 2007 Dec;7:1-5.
31. Farnia P, Mohammadi F, Masjedi MR, Varnerot A, Zarifi AZ, Tabatabee J, Douraghei M, Ghazisaeedi K, Mansorri D, Bahadori M, Vincent V. Evaluation of tuberculosis transmission in Tehran: using RFLP and spoloigotyping methods. Journal of Infection. 2004 Aug 1;49(2):94-101.
32. Doroudchi M, Kremer K, Basiri EA, Kadivar MR, Van Soolingen D, Ghaderi AA. IS6110-RFLP and spoligotyping of Mycobacterium tuberculosis isolates in Iran. Scandinavian journal of infectious diseases. 2000 Jan 1;32(6):663-8.
33. Small PM, Shafer RW, Hopewell PC, Singh SP, Murphy MJ, Desmond ED, Sierra MF, Schoolnik GK. Exogenous reinfection with multidrug-resistant Mycobacterium tuberculosis in patients with advanced HIV infection. New England Journal of Medicine. 1993 Apr 22;328(16):1137-44.