[Detection of rifampin resistant mycobacterium tuberculosis complex using denaturing HPLC].
BACKGROUND
Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence.


METHODS
In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing.


RESULTS
Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing.


CONCLUSIONS
DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.
Nam
Youn Hyoung
YH
Departmnt of Chemistry, School of Advanced Science and Basic Science Research Institute Dankook University, Cheonan, Korea.
Lee
Sang Hyun
SH
Ahn
Young Chang
YC
Cho
Min Ho
MH
Jang
Won Cheoul
WC
Park
Su Min
SM
Kwon
Pil Seung
PS
Kim
Jong Wan
JW
D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D015373:Bacterial Typing Techniques; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004269:DNA, Bacterial; D024881:Drug Resistance, Bacterial / Q000235:genetics; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D012293:Rifampin / Q000494:pharmacology*; D014376:Tuberculosis / Q000382:microbiology*
D000904:Antibiotics, Antitubercular; D004269:DNA, Bacterial; D012293:Rifampin