Capillary Electrophoresis-based Heteroduplex Analysis with a Universal Heteroduplex Generator for Detection of Point Mutations Associated with Rifampin Resistance in Tuberculosis

Abstract

Background: Slab gel heteroduplex analysis (HDA), a popular scanning method for genetic mutations, uses DNA fragments typically generated by PCR to create homo- and heteroduplex molecules with conformational differences and sequence-dependent electrophoretic profiles. Use of a universal heteroduplex generator (UHG) enhances the subtle variations caused by single-base substitutions. Methods: The HDA-UHG slab gel format was modified for an efficient capillary-based method. The effect of staining dyes TOPRO5 and YOPRO1 on the analysis of heteroduplexes was studied, as well as ultraviolet absorbance and laser-induced fluorescence (LIF) detection methods. In addition, the entangled polymers hydroxyethyl cellulose, methyl cellulose, and linear polyacrylamide were evaluated as separation matrices. Results: This assay was able to detect the presence of Mycobacterium tuberculosis and its rifampin susceptibility directly from clinical specimens in dramatically reduced analysis time (30 min vs 2.5 h). Optimized conditions included 0.3% methyl cellulose as the separation matrix, on-line staining using 1 μmol/L YOPRO1, and LIF detection for quantitative and reproducible analysis of single-base substitutions in the rifampin resistance-determining region of rpoB that give rise to the rifampin-resistant phenotype of M. tuberculosis. We generated 95% confidence limits using the wild-type sequence and used these limits to determine rifampin susceptibility in samples. Conclusions: Capillary electrophoresis, combined with the HDA-UHG technique, may be of value for rapid and efficient clinical diagnosis of rifampin-resistant tuberculosis strains.

Biosensor-based detection of tuberculosis

“…44 A hetero-duplex analysis method has also been developed in recent years where strands with a single-base mismatch can easily be identied and separated utilizing conformation-sensitive gel electrophoresis from the strands containing no mismatches. 45,46 (b) Hybridization-based techniques: this type of technique relies on the hybridization of clinical DNA with complementary DNA and the binding is then compared with the results of wild/ reference M. tb. DNA.…”

Capillary Electrophoresis for the Analysis of Biopolymers

“…These intramolecular bonds are sensitive to the composition of the DNA, and a single-base polymorphism often generates a detectable mobility shift (177). A universal heteroduplex generator was used to monitor point mutations associated with rifampin resistance in Mycobacterium tuberculosis by capillary electrophoresis, with a significant improvement in analysis time (178).…”

Determination of DNA methylation by COBRA: A comparative study of CGE with LIF detection and conventional gel electrophoresis

“…Herein, we report on a comparison of this method with a commonly used slab‐gel electrophoresis method. Other groups from different fields of research have already examined the separation of DNA by gel electrophoresis in contrast to CGE 17–23, albeit from other points of interest. Only Beaubier et al .…”