Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis

Very fast amplification of DNA in small volumes can be continuously monitor ed with a rapid cycler that incorporates fluorimetric detection. Primers we re designed to amplify a 157-bp fragment of the rpoB gene spanning codons 5 26 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Myc obacterium tuberculosis. Most mutations associated with resistance to rifam pin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. T wo pairs of hybridization probes were synthesized; one in each pair was 3' labeled with fluorescein and hybridized upstream of the codon with the muta tion; the other two probes were 5' labeled with LightCycler-Red 640, Each p air of probes recognized adjacent sequences in the amplicon, After DNA ampl ification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt progr am, Twenty M, tuberculosis clinical isolates susceptible to streptomycin, I NH, RMP, and ethambutol and 36 antibiotic-resistant clinical M, tuberculosi s isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial age nts) were amplified, and the presence of mutations was determined using sin gle-strand conformation polymorphism analysis, the LiQor automated sequence r, and the LightCycler system. Concordant results were obtained in all case s. Within 30 min, the LightCycler method correctly genotyped all the strain s without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-a ssociated mutations in M. tuberculosis clinical isolates.