Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during treatment of tuberculosis

Abstract

Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of M. tuberculosis DNA quantification were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantification with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of M. tuberculosis DNA quantified in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB, M. tuberculosis DNA, and cultivable bacilli were similar, suggesting that quantification of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and M. tuberculosis DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efficacy.

FIG. 1

Sequence of the region of M. tuberculosis IS6110 amplified in both competitive and TaqMan PCRs. The primers for competitive PCR are labeled T4 and T5. The primers used in the ABI 7700 system are labeled IS6 and IS7. The BglI site is the site of insertion of the 80-bp M. avium fragment to make the internal control plasmid pQIS6110.

FIG. 2

Competitive PCRs for M. bovis genomic DNA and pIS6110 plasmid DNA. (A) Autoradiograph of two competitive PCRs. PCR amplification of the internal control plasmid, pQIS6110, results in a 203-bp product. Amplification of test DNAs from both M. bovis and pIS6110 results in a 123-bp product. The number of pQIS6110 molecules added to each PCR mixture is listed under the lane. (B) Relative amounts of sample and internal control DNA for the data in the M. bovis gel in panel A. The equation and fit of the line are shown.

FIG. 3

Standard curves generated by analysis of known amounts of template DNA with the ABI 7700 system. Purified DNA from M. bovis 410, plasmid pIS6110, and internal control plasmid pQIS6110, each containing one copy of the IS6110 insertion element, were assayed in parallel. The regression lines calculated for the datum points are shown. The R² values for each of the lines was >0.99.

FIG. 4

Comparison of TaqMan and competitive PCR assays for two patients receiving tuberculosis chemotherapy. IS6110 DNA levels were determined with both assay systems for DNA isolated from sputum collected during the course of chemotherapy. The same DNA sample was tested in both systems. Specimens were collected in triplicate on day 0, before the onset of antimicrobial chemotherapy, and duplicate samples were collected thereafter. The competitive assay was performed once per sample. Error bars show the standard deviations for duplicate TaqMan measurements.

FIG. 5

Amounts of AFB, numbers of CFU, and IS6110 DNA levels in sputum collected during the first 2 to 3 months of chemotherapy. Data for two patients are shown. Values represent the highest measurement for replicate specimens collected on a given day.