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 1

Keywords

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

ASJC Scopus subject areas

  • Bioengineering
  • Biomedical Engineering
  • Molecular Biology

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