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Journal of Clinical Microbiology, July 2001, p. 2531-2540, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2531-2540.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification of Rifampin-Resistant Mycobacterium tuberculosis Strains by Hybridization, PCR, and Ligase Detection Reaction on Oligonucleotide Microchips

Vladimir Mikhailovich,1 Sergey Lapa,1 Dimitry Gryadunov,1 Alexander Sobolev,1 Boris Strizhkov,1 Nikolai Chernyh,1 Olga Skotnikova,2 Olga Irtuganova,2 Arkadii Moroz,2 Vitalii Litvinov,2 Mikhail Vladimirskii,3 Mikhail Perelman,3 Larisa Chernousova,4 Vladislav Erokhin,4 Alexander Zasedatelev,1 and Andrei Mirzabekov1,5,*

Biochip Technology Center, Argonne National Laboratory, Argonne, Illinois,5 and Moscow Anti-Tuberculosis Center, Moscow Government,2 Research Institute for Phthisiopulmonology, I. M. Sechenov Moscow Medical Academy,3 Engelhardt Institute of Molecular Biology,1 and Central TB Research Institute, Russian Academy of Medical Sciences,4 Moscow, Russia

Received 22 December 2000/Returned for modification 21 February 2001/Accepted 21 April 2001

Three new molecular approaches were developed to identify drug-resistant strains of Mycobacterium tuberculosis using biochips with oligonucleotides immobilized in polyacrylamide gel pads. These approaches are significantly faster than traditional bacteriological methods. All three approaches---hybridization, PCR, and ligase detection reaction---were designed to analyze an 81-bp fragment of the gene rpoB encoding the beta -subunit of RNA polymerase, where most known mutations of rifampin resistance are located. The call set for hybridization analysis consisted of 42 immobilized oligonucleotides and enabled us to identify 30 mutant variants of the rpoB gene within 24 h. These variants are found in 95% of all mutants whose rifampin resistance is caused by mutations in the 81-bp fragment. Using the second approach, allele-specific on-chip PCR, it was possible to directly identify mutations in clinical samples within 1.5 h. The third approach, on-chip ligase detection reaction, was sensitive enough to reveal rifampin-resistant strains in a model mixture containing 1% of resistant and 99% of susceptible bacteria. This level of sensitivity is comparable to that from the determination of M. tuberculosis drug resistance by using standard bacteriological tests.

* Corresponding author. Mailing address: Biochip Technology Center, Engelhardt Institute of Molecular Biology, 32 Vavilova St., Moscow 119991, GSP-1, Russia. Phone: 7 (095) 135 0559. Fax: 7 (095) 135 1405. E-mail: amir{at}genome.eimb.relarn.ru.

Journal of Clinical Microbiology, July 2001, p. 2531-2540, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2531-2540.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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