サルモネラ特異的PCR産物のDNA固定化水晶振動子による検出

Translated title of the contribution: Rapid Detection Method of Salmonella-specific PCR Product by DNA-immobilized Quartz Crystal Microbalance.

宮本 敬久, Sudsai Trevanich, 岡部 隆史, 友田 諭, 本城 賢一, 波多野 昌二

Research output: Contribution to journalArticle

Abstract

The use of DNA-immobilized quartz crystal microbalance (QCM) for rapid detection of <I>Salmonella</I> has been demonstrated. The terminal 5'-phosphate group of a DNA fragment containing <I>Salmonella</I>-specific 464 bp DNA was modified with ethylendiamine. The DNA was denatured by heating and covalently immobilized via glutaraldehyde on a QCM treated with 3-aminopropyltriethoxysilane. The QCM immobilized with a single stranded DNA fragment containing the <I>Salmonella</I>-specific DNA was connected to Sogo Pharmaceutical Fragrance sensor SF-105W. The frequency of the QCM decreased by the addition of complementary single strand DNA. The frequency also decreased after incubation with the PCR reaction mixture from <I>Salmonella</I>-genomic DNA and du primer. However, the frequency did not decrease after incubation with phage DNA, plasmid DNA or PCR reaction mixtures from <I>E. coli</I> and <I>B. cereus</I>. The frequency change of the QCM after incubation with complementary DNA was closely correlated to the amount of DNA in the sample. The correlation coefficient was 0.995. Strand separation of the DNA-DNA hybrid at the surface of QCM was achieved by exposure to 10mM NaOH. This allowed the immobilized 464-mer DNA to be reused. Chicken meat inoculated with <I>Salmonella</I> Typhimurium IFO 12529 at 108 CFU/25g was cultured in EEM broth for 6h and selectively enriched in DMPBN medium for 18h. DNA was prepared from the culture and PCR was performed with the du primer. Using this <I>Salmonella</I>-specific DNA immobilized QCM, a decrease in frequency was detected with the PCR reaction mixture.
Original languageJapanese
Pages (from-to)57-63
Number of pages7
Journal日本食品微生物学会雑誌 = Japanese journal of food microbiology
Volume16
Issue number1
DOIs
Publication statusPublished - 1999

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Immobilized Nucleic Acids
Salmonella
Quartz crystal microbalances
DNA
Fragrances
Bacteriophages
Meats
Single-Stranded DNA
Glutaral

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サルモネラ特異的PCR産物のDNA固定化水晶振動子による検出. / 宮本敬久; Trevanich, Sudsai; 岡部隆史; 友田諭; 本城賢一; 波多野昌二.

In: 日本食品微生物学会雑誌 = Japanese journal of food microbiology, Vol. 16, No. 1, 1999, p. 57-63.

Research output: Contribution to journalArticle

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abstract = "The use of DNA-immobilized quartz crystal microbalance (QCM) for rapid detection of <I>Salmonella has been demonstrated. The terminal 5'-phosphate group of a DNA fragment containing <I>Salmonella-specific 464 bp DNA was modified with ethylendiamine. The DNA was denatured by heating and covalently immobilized via glutaraldehyde on a QCM treated with 3-aminopropyltriethoxysilane. The QCM immobilized with a single stranded DNA fragment containing the <I>Salmonella-specific DNA was connected to Sogo Pharmaceutical Fragrance sensor SF-105W. The frequency of the QCM decreased by the addition of complementary single strand DNA. The frequency also decreased after incubation with the PCR reaction mixture from <I>Salmonella-genomic DNA and du primer. However, the frequency did not decrease after incubation with phage DNA, plasmid DNA or PCR reaction mixtures from <I>E. coli and <I>B. cereus. The frequency change of the QCM after incubation with complementary DNA was closely correlated to the amount of DNA in the sample. The correlation coefficient was 0.995. Strand separation of the DNA-DNA hybrid at the surface of QCM was achieved by exposure to 10mM NaOH. This allowed the immobilized 464-mer DNA to be reused. Chicken meat inoculated with <I>Salmonella Typhimurium IFO 12529 at 108 CFU/25g was cultured in EEM broth for 6h and selectively enriched in DMPBN medium for 18h. DNA was prepared from the culture and PCR was performed with the du primer. Using this <I>Salmonella-specific DNA immobilized QCM, a decrease in frequency was detected with the PCR reaction mixture.",
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