リアルタイム PCR による納豆中の黄色ブドウ球菌 …

1 14 6 10 2005 6 PCR Rapid Detection of Staphylococcus aureus in Natto Using Real-time PCR To moh iro Hosoi Fermented soy foods made with Bacillus subtilis, such as natto, are produced in Japan and several other East Asian countries. During the manufacturing process, components of these products and their containers are touched or exposed to the environment, which causes contamination with other microorganisms. In this study, we attempted to develop a method for detecting a small number of Staphylococcus aureus in natto using the LightCycler real-time PCR system. By real-time PCR with a primer set for the nuc gene after an enrichment culture with Giolitti-Cantoni broth at 35 C for 20 h, S. aureus at a concentration of more than 12 CFU/30 g (in natto) was detected.

2 (Accepted Mar. 10, 2005) Bacillus subtilis natto dou chi kinema tua nao chungkuk-jang 1) 1 B. subtilis natto 104 CFU/g Staphylococcus aureus nuclease nuc 2,3,4,5)

3 PCR Polymerase chain reaction 14 2005 Giolitti-Cantoni 1. Escherichia coli O157 188-4 Staphylococcus warneri 15 Japan Collection of Microorganisms JCM BBL Becton Dickinson and Company 30 C 48 2 12000 g 10 2. nuc 2,3,4,5) 16S rDNA 5' 350 6) nuc nuc DNA Data Bank of Japan Accession Number V01281 Primer Express Version PCR 1 DNA DNA Isoplant II DNeasy Plant Mini Kit DNA Gene Spec III 260nm g/ L PCR PCR PCR QuantiTect SYBR Green PCR DNA 2 L 10 pmol/ L 1 L 20 L PCR 95 C 15 94 C 15 56 C 30 72 C 50 50 LightCycler Software version 65 C 95 C C/ SYBR Green I nucleic acid gel stain BMA Agarose Mupid-21

4 1 (5'-3') (bp) nuc sense AGGGATGGCTATCAGTAATG 114 antisense GCTGAGCTACTTAGACTTGAAA 16S rDNA sense GAGTTTGATCCTGGCTCAG 349* antisense CTGCTGCCTCCCGTAG *Escherichia coli (position 9-357) 3. Bacillus subtilis JCM 1465 105 CFU/mL 107 CFU/mL Staphylococcus aureus JCM 2413 102 CFU/mL 107 CFU/mL DNA 2 L PCR 70 PCR 4. 109 CFU/g 30g Potassium tellurite trihydrate Giolitti-Cantoni 50mL 0 109 CFU/30 g 35 C 20 PCR PCR 95 C 15 94 C 15 56 C, 40 72 C 50 70 PCR DNA 3 1 mL 12000 g 10 100 L 10 10 12000 g 10 DNA 7 1.

5 Nuclease 100 C 1 DNA RNA 4) nuc 16 DNA PCR 50 1 13 S. aureus JCM 2413 75 C PCR DNA 16S rDNA PCR 2 nuc 12 S. aureus JCM 2413 16S rDNA8 1, Water; 2, Enterobacter aerogenes JCM 1235; 3, Escherichia coli JCM 1649; 4, Escherichia coli O157 188-4; 5, Klebsiella pneumoniae JCM 1662; 6, Salmonella choleraesuis JCM 1652; 7, Vibrio fluvialis JCM 3752; 8, Pseudomonas aeruginosa JCM 5516; 9, Pseudomonas fluorescens JCM 5963; 10, Flavobacterium odoratum JCM 7458; 11, Bacillus subtilis JCM 1465; 12, Bacillus cereus JCM 2152; 13, Staphylococcus aureus JCM 2413; 14, Staphylococcus epidermidis JCM 2414; 15, Enterococcus faecalis JCM 5803; 16, Enterococcus faecium JCM 5804; 17, Micrococcus luteus JCM 1464 1 DNA nuc LightCycler real-time PCR 1, Enterobacter aerogenes JCM 1235; 2, Escherichia coli JCM 1649; 3, Escherichia coli O157 188-4; 4, Klebsiella pneumoniae JCM 1662.

6 5, Salmonella choleraesuis JCM 1652; 6, Vibrio fluvialis JCM 3752; 7, Pseudomonas aeruginosa JCM 5516; 8, Pseudomonas fluorescens JCM 5963; 9, Flavobacterium odoratum JCM 7458; 10, Bacillus subtilis JCM 1465; 11, Bacillus cereus JCM 2152; 12, Staphylococcus aureus JCM 2413; 13, Staphylococcus epidermidis JCM 2414; 14, Enterococcus faecalis JCM 5803; 15, Enterococcus faecium JCM 5804; 16, Micrococcus luteus JCM 1464; M, 100bp DNA ladder Marker (Takara Bio) 2 DNA nuc 16S rDNA real-time PCR 14 2005 PCR S. epidermidis S. warneri nuc Brakstad nuc PCR 90 S. aureus 16 80 100% 3) nuclease S.

7 Aureus S. intermedius S. hyicus S. schleiferi 3, 5) nuc S. aureus JCM 2413 1 2. 105 CFU/mL 107 CFU/mL 3 3. Giolitti-Cantoni PCR 35 C 20 PCR 12 CFU/30 g 4 108 CFU/30 g PCR 12 CFU/30g 9 3 real-time PCR 4 real-time PCR Bacillus subtilis nuc PCR

8 Giolitti-Cantoni 35 C 20 12 CFU/30 g PCR 1) Hosoi, T. and Kiuchi, K., Production and probiotic effects of natto. In Bacterial spore formers, eds. Ricca, E., Henriques, , and Cutting, Horizon Bioscience, pp. 143-153 (2004). 2) Shortle, D., A genetic system for analysis of staphylococcal nuclease. Gene, 22, 181-189 (1983). 3) Brakstad, , Aasbakk, K., and Maeland, , Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol, 30, 1654-1660 (1992). 4) Lachica, , Hoeprich, , and Riemann, , Tolerance of staphylococcal thermonuclease to stress. Appl. Microbiol, 23, 994-997 (1972). 5) Gudding, R., Differentiation of staphylococci on the basis of nuclease properties.

9 J. Clin. Microbiol, 18, 1098-1101 (1983). 6) pp. 83-132 2001 . 17 3 10 10