Simultaneous detection of multiple mutations conferring streptomycin resistance inMycobacterium tuberculosis using nanoscale engineered biomagnetites

Kohei Maruyama, Norikuni Uchida, Haruko Takeyama, Tetsushi Mori, Ryuji Kawaguchi, Tadashi Matsunaga

    Research output: Contribution to journalArticle

    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.

    Original languageEnglish
    Pages (from-to)71-78
    Number of pages8
    JournalNanobiotechnology
    Volume2
    Issue number3-4
    DOIs
    Publication statusPublished - 2006 Sep

    Fingerprint

    Streptavidin
    Streptomycin
    Tuberculosis
    Genes
    Mutation
    DNA
    Cost effectiveness
    Biotin
    Nucleotides
    Buffers
    Salts
    Polymerase Chain Reaction
    Point Mutation
    Cost-Benefit Analysis
    Hot Temperature
    Temperature

    Keywords

    • Automated system
    • Bacterial magnetic particles (BacMPs)
    • Mycobacterium tuberculosis
    • Nanoscale-engineered biomagnetite
    • Simultaneous detection of multiple mutations
    • Streptomycin-resistant mutations

    ASJC Scopus subject areas

    • Molecular Biology
    • Bioengineering
    • Biomedical Engineering

    Cite this

    Simultaneous detection of multiple mutations conferring streptomycin resistance inMycobacterium tuberculosis using nanoscale engineered biomagnetites. / Maruyama, Kohei; Uchida, Norikuni; Takeyama, Haruko; Mori, Tetsushi; Kawaguchi, Ryuji; Matsunaga, Tadashi.

    In: Nanobiotechnology, Vol. 2, No. 3-4, 09.2006, p. 71-78.

    Research output: Contribution to journalArticle

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