Simultaneous detection of multiple mutations conferring streptomycin resistance inMycobacterium tuberculosis using nanoscale engineered biomagnetites |
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Authors: | Kohei Maruyama Norikuni Uchida Haruko Takeyama Tetsushi Mori Ryuji Kawaguchi Tadashi Matsunaga |
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Institution: | (1) Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, 184-8588 Tokyo, Japan;(2) PropGene Inc., 2-24-16 Naka-cho, Koganei, 184-8588 Tokyo, Japan;(3) Institute for Biomedical Engineering, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, 162-0041 Tokyo, Japan |
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Abstract: | Streptomycin-resistantMycobacterium tuberculosis has been attributed to two distinct classes of mutations, including point mutations within therpsL gene (three mutation sites) and therrs gene (seven mutation sites). We have developed an automated simultaneous detection system of multiple mutations based on
thermal dissociation curve analysis for streptomycin resistance inM. tuberculosis using streptavidin-labeled bacterial magnetic particles (SA-BacMPs). With consideration for time and cost effectiveness,
we used fewer PCR reactions, with a long PCR target (rpsL, 182 bp;rrs, 467 bp) including multiple mutation sites. In order to improve the amount of target DNA captured on BacMPs through streptavidin-biotin
binding, several reaction conditions, such as salt species and concentration in the buffer, and reaction temperature were
examined. Compared to the commonly used 1M NaCl solution, the amount of DNA captured on SA-BacMPs was about six times greater (approx 5 pmoles/50 μg BacMPs) in the
2M LiCl solution. Under these conditions, automated nucleotide discriminations of 10 targets inrpsL andrrs genes of streptomycin-resistant and wild-type strains were successfully performed at the same time. |
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Keywords: | Nanoscale-engineered biomagnetite bacterial magnetic particles (BacMPs) streptomycin-resistant mutations Mycobacterium tuberculosis simultaneous detection of multiple mutations automated system |
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