[Rapid detection of rifampin resistance of Mycobacterium tuberculosis using high-throughput pyrosequencing technique].
OBJECTIVE
To develop a pyrosequencing approach to rapid detection of rifampin resistance in Mycobacterium tuberculosis based on characterization of all possible mutations in the 81 bp core region.


METHODS
Two pyrosequencing sequencing primers and 1 pair of PCR primers were chosen for pyrosequencing analysis. The sensitivity of the pyrosequencing approach was determined by assaying PCR products generated from 10-fold serial dilutions of the DNA from Mycobacterium tuberculosis H(37) Rv strains. The efficacy of the pyrosequencing approach was evaluated by analyzing clinical Mycobacterium tuberculosis isolates with known antibiotic phenotypes.


RESULTS
Rifampin resistance could be determined within 2 hours after PCR amplification and single-stranded template preparation by using only two pyrosequencing reactions. About 50 fg DNA/reaction was required in order to get sufficient PCR product for producing a long, clear and accurate pyrosequencing pattern. A total of 41 Mycobacterium tuberculosis isolates were tested and the results were concordant with those based on drug susceptibility testing and conventional DNA sequencing.


CONCLUSIONS
The pyrosequencing technique is simple to perform and can be used as a rapid, high-throughput and efficient method for detecting rifampin resistance in Mycobacterium tuberculosis.
Zhao
Jin-rong
JR
Institute of Genetic Diagnosis, Department of Pharmacology, Fourth Military Medical University, Xi'an 710032, China.
Bai
Yu-jie
YJ
Wang
Yan
Y
Zhang
Qing-hua
QH
Luo
Ming
M
Yan
Xiao-jun
XJ
D000995:Antitubercular Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012293:Rifampin / Q000494:pharmacology*; D017422:Sequence Analysis, DNA / Q000379:methods*
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D012293:Rifampin