Development of a pyrosequencing approach for rapid screening of rifampin, isoniazid and ethambutol-resistant Mycobacterium tuberculosis.
METHODS
The need to minimize the transmission of drug-resistant Mycobacterium tuberculosis requires rapid identification procedures.


OBJECTIVE
To develop a pyrosequencing approach for rapid screening of rifampin, isoniazid and ethambutol-resistant M. tuberculosis based on characterization of resistance-associated hot mutations.


METHODS
Three pairs of PCR primers and three pyrosequencing sequencing primers for detecting mutations at codon 526 and 531 of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene were chosen. The sensitivity of the pyrosequencing approach was determined by assaying PCR products generated from 10-fold serial dilutions of the DNA from the H37Rv strain. The efficacy of the pyrosequencing approach was evaluated by analyzing clinical isolates with a known antibiotic phenotype.


RESULTS
Resistance-associated hot mutations could be determined within 2 h after PCR amplification using pyrosequencing. About 45 fg DNA per reaction was required to obtain sufficient PCR products to produce a clear, accurate pyrosequencing pattern. No mutations were found in all 20 drug-susceptible clinical isolates, while all isolates with mutations showed corresponding drug resistances.


CONCLUSIONS
This pyrosequencing approach can be used for rapid screening of rifampin-, isoniazid- and ethambutol-resistant M. tuberculosis prior to standard drug susceptibility testing.
Zhao
J-R
JR
Institute of Genetic Diagnosis, Fourth Military Medical University, Xi'an, China.
Bai
Y-J
YJ
Wang
Y
Y
Zhang
Q-H
QH
Luo
M
M
Yan
X-J
XJ
D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D003062:Codon; D004252:DNA Mutational Analysis / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis; D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D004977:Ethambutol / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007538:Isoniazid / Q000494:pharmacology; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*
D000904:Antibiotics, Antitubercular; D003062:Codon; D004269:DNA, Bacterial; D004977:Ethambutol; D007538:Isoniazid; D012293:Rifampin