B. Afghani et Hr. Stutman, QUANTITATIVE-COMPETITIVE POLYMERASE CHAIN-REACTION FOR RAPID SUSCEPTIBILITY TESTING OF MYCOBACTERIUM-TUBERCULOSIS TO ISONIAZID, Biochemical and molecular medicine, 60(2), 1997, pp. 182-186
The objective of this study was to determine whether quantitative-comp
etitive polymerase chain reaction (QC-PCR) can be used for rapid susce
ptibility testing of Mycobacterium tuberculosis (MTB). QC-PCR was used
to determine relative amounts of mycobacterial DNA inoculated at diff
erent isoniazid (INN) concentrations, A total of sig different INH-sen
sitive (INH-S) and five INH-resistant (INH-S) strains were inoculated
in the presence of 8.0, 0.2, 1.0, and 10.0 mu g/ml of INH. DNA was qua
ntified 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 greater than or equal to 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 iso
lates there were no apparent differences in DNA concentration between
the control suspensions and those containing 0.2 and 1.0 mu g/ml INH d
uring the 3-week incubation period. The highest INH concentration (10
mu g/ml), however, did abolish the DNA increase seen in the other MTB
suspensions. This preliminary study suggests that by using concentrati
ons of 0.2 or 1.0 mu g/ml of INN, QC-PCR may differentiate INN-R and I
NH-S MTB isolates within 1 week. This method may be of particular valu
e when applied directly to clinical specimens with varying numbers of
bacilli. (C) 1997 Academic Press.