Single Tube Single-Color Analysis Of Rifampicin And Isoniazid Allelic Variants In Mdr-Tb

Background: We have constructed a highly informative, rapid, single-tube, single-color assay for distinguishing variants in the inhA promotor, and katG and rpoB gene targets responsible for isoniazid and rifampicin resistance, using technologies invented at Brandeis University. Methods & Materials: A multiplex LATE-PCR reaction is used to generate single-stranded DNA products for the Rifampicin Resistance Determining Region of the rpoB gene, the katG gene, and the inhA promotor, as well as an amplifiable internal control. These four amplicons are detected with four sets of Therma-Light probes all of which are labeled with Black Hole Quenchers with/without Quasar 670 fluorophors. Probe-target hybridization takes place at end-point over a wide range of temperatures below the reaction annealing temperature. The temperature ranges used for the four sets of Therma-Light probes are deliberately overlapped, making it possible to maximize the amount of information generated in a single color. The amplifiable internal control generates a specific very low temperature signal and an additional non-amplifiable internal control generates a second signal at a specific high temperature. Together these controls can be used to calibrate each reaction. Another proprietary reagent is added to the reaction mixture to improve primer specificity. Results: The present assay has been tested on twenty-two different archival strains which are reported to harbor many of the common alleles responsible for resistance to rifampin and isoniazid. Each strain displayed its own “fluorescent signature” reflecting the underlying alleles present in the three target sequences. But, not every fluorescent signature agreed with archival information on the mutational composition of the strain. These discrepancies were resolved by sequencing the reaction products. The results show that the fluorescent signatures consistently identify the alleles correctly. Many more strains of M. tuberculosis will soon be tested. Conclusion: This single color multiplex assay has both clinical and research applications and is just one of many possible assays for M(X)DR-TB that can be constructed using these technologies. For instance, Therma-Light probes labeled with fluorophores in other colors can be added for simultaneous analysis variants responsible for resistance to all other first and second line antibiotics. Support: Brandeis University and Hain Lifescience