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Background  DNA fingerprinting by IS 6110 -RFLP has shown a high incidence of  Mycobacterium tuberculosis  isolates having no and low copies of the insertion sequence in Kerala, South India. Amplified Fragment Length Polymorphism (AFLP) would scan the entire genome rather than a few repetitive elements, we thought that this technique would help us in differentiating the large reservoir of isolates from an endemic region. Here we evaluate the ability of Amplified Fragment Length Polymorphism (AFLP) to type clinical isolates.    Methods  Fifty clinical isolates of  M. tuberculosis  were analysed by conventional radioactive AFLP and IS 6110-  RFLP.  M. bovis ,  M. bovis  BCG and two non tuberculous mycobacteria were also analysed to see species specific differences generated by AFLP. Cluster analysis was performed using the AFLP profile that showed the maximum polymorphism within  M. tuberculosis  and this was compared to the number of copies of IS 6110  insertions.    Results  For AFLP, out of ten primer pairs tested, the EO/MC pair generated maximum polymorphism among the clinical isolates of  M. tuberculosis . The similarity between the isolates ranged between 88 and 99.5%. Majority (nearly 85%) of the 'low copy' IS 6110  isolates clustered together, while the rest clustered irrespective of the copy numbers. AFLP could show rare differences between isolates of  M. tuberculosis ,  M. bovis  and  M. bovis  BCG. The AFLP profiles for non-tuberculous mycobacteria were highly different from those of  M. tuberculosis .    Conclusion  Polymorphism generated by AFLP within the  M. tuberculosis  species is limited and hence AFLP alone seems to have limited use in fingerprinting the isolates in Kerala. The combined use of AFLP and IS 6110 -RFLP showed relatively better differentiation of 'high copy' IS 6110  isolates, but failed to differentiate the 'low copy' isolates. However, the technique may be efficient in inter-species differentiation, and hence potentially useful in identifying and developing species- specific markers.

bmc infectious diseases

Combined use of Amplified Fragment Length Polymorphism and IS6110 -RFLP in fingerprinting clinical isolates of Mycobacterium tuberculosis from Kerala, South India