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Clinical Chemistry 47: 1195-1203, 2001;
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(Clinical Chemistry. 2001;47:1195-1203.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

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

Gloria A. Thomas1, Diana L. Williams2 and Steven A. Soper1a

1 Chemistry Department, Louisiana State University, Baton Rouge, LA 70803.
2 Molecular Biology Research Department, Laboratory Research Branch, National Hansen’s Disease Programs at the School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803.


aAuthor for correspondence. Fax 225-388-3458; e-mail steve.soper{at}chem lsu.edu

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.




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Microarray-based pncA genotyping of pyrazinamide-resistant strains of Mycobacterium tuberculosis
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Fluorescent Heteroduplex Assay for Monitoring Bacillus anthracis and Close Relatives in Environmental Samples
Appl. Envir. Microbiol., June 1, 2003; 69(6): 3317 - 3326.
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