Quantitative-competitive polymerase chain reaction for rapid susceptibility testing of Mycobacterium tuberculosis to isoniazid

Abstract

The objective of this study was to determine whether quantitative-competitive polymerase chain reaction (QC-PCR) can be used for rapid susceptibility testing of Mycobacterium tuberculosis (MTB). QC-PCR was used to determine relative amounts of mycobacterial DNA inoculated at different isoniazid (INH) concentrations. A total of six different INH-sensitive (INH-S) and five INH-resistant (INH-R) strains were inoculated in the presence of 0.0, 0.2, 1.0, and 10.0 micrograms/ml of INH. DNA was quantified using QC-PCR at Week 0 and weekly thereafter for 3 weeks. For the QC-PCR, 10-fold dilutions of control (240 bp) DNA having the same primer set as the target DNA (123 bp) were used. The amount of target DNA was estimated by using known amounts of the internal standard. For INH-S isolates there was > or = 1 log difference in DNA concentration in the presence of each INH concentration compared to that of the control within 1 to 3 weeks. In contrast, for INH-R isolates there were no apparent differences in DNA concentration between the control suspensions and those containing 0.2 and 1.0 microgram/ml INH during the 3-week incubation period. The highest INH concentration (10 micrograms/ml), however, did abolish the DNA increase seen in the other MTB suspensions. This preliminary study suggests that by using concentrations of 0.2 or 1.0 microgram/ml of INH, QC-PCR may differentiate INH-R and INH-S MTB isolates within 1 week. This method may be of particular value when applied directly to clinical specimens with varying numbers of bacilli.