COMPARATIVE-EVALUATION OF CLEAVASE FRAGMENT LENGTH POLYMORPHISM WITH PCR-SSCP AND PCR-RFLP TO DETECT ANTIMICROBIAL AGENT RESISTANCE IN MYCOBACTERIUM-TUBERCULOSIS

Background: Several molecular methods potentially useful in the detect ion of Mycobacterium tuberculosis mutations, specifically in rpoB and katG, were compared. Methods and Results: DNA from 24 M. tuberculosis clinical isolates, with mutations associated with resistance to rifamp in and/or isoniazid, was analyzed. A 128 bp amplicon, spanning the 81 bp rpoB region containing most mutations leading to rifampin resistanc e, was analyzed by polymerase chain reaction-single-strand conformatio n polymorphism (PCR-SSCP) and a recently introduced mutation scanning method, cleavase fragment length polymorphism (CFLP) analysis. Also, a 350 bp amplicon encompassing that region was analyzed by the CFLP met hod. CFLP analysis of the 350 bp amplicon (23 isolates) identified 14 of 17 mutants; however? CFLP analysis of the 128 bp amplicon accuratel y identified all mutants as did PCR-SSCP with interpretative difficult y for two codon 513 mutations. CFLP and PCR-restriction fragment lengt h polymorphism (RFLP) analyses of a 623 bp amplicon encompassing katG codons 315 and 463 showed that the CFLP method identified single and d inucleotide codon 315 substitutions with or without codon 463 (CGG-->C TG) changes, whereas PCR-RFLP (MspI) missed one codon 315 polymorphism (AGC-->ACA) in three isolates. Conclusion: Both PCR-SSCP and CFLP ana lyses were sensitive in identifying all mutations on short sequences i n the rpoB mutants. CFLP appears to be more efficient than SSCP and RF LP for the detection of mutations in large amplicons.