緒言 Listeria monocytogenes ready-to-eat L. monocytogenes ISO - FDA/BAM L. monocytogenes Real-Time PCR DNA PCR SYBR Green ds-dna PCR LAMP Molecular Dete

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1 Rep. Nat l Food Res. Inst No 報文 3M TM Molecular Detection System を用いた Listeria monocytogenes の簡易迅速遺伝子検査法の評価 Evaluation of 3M Molecular Detection System for detecting Listeria monocytogenes from food samples Susumu KAWASAKI 1 Mari Mochida 1, Mie Saito 1, and Takatoshi Moriyama 2 1 National Food Research Institute, Kannondai, Tsukuba, Ibaraki , Japan 2 3M Health Care Limited, 33-1, Tamagawadai 2-chome Setagaya-ku, Tokyo , Japan Abstract The culture methods (ISO ) were compared to 3M Molecular detection system (MDS) for Listeria monocytogenes from enrichment cultures of various types of artificially inoculated ready to eat (RTE) foods. The MDS assay was evaluated in 12 types of spiked samples, including meat, fish, sprouts, and dairy products for L. monocytogenes. The MDS assay also evaluated in inoculate ground beef samples. Beef samples contamination levels is 0.66 most probable number (MPN)/g were used for recovery study. Twenty test portions (5g) of the contaminated beef were pre-enriched, and MDS assay and the culture method (ISO ) was used thereafter. An excellent agreement was obtained for the results of MDS assay and culture method, which suggests that the MDS assay is a reliable and useful method for rapid screening of L. monocytogenes contamination. Keywords: 3M Molecular detection system, L. monocytogenes, rapid detection, foodborne pathogens Corresponding author skawasa@affrc.go.jp

2 緒言 Listeria monocytogenes ready-to-eat L. monocytogenes ISO - FDA/BAM L. monocytogenes Real-Time PCR DNA PCR SYBR Green ds-dna PCR LAMP Molecular Detection System MDS LAMP ATP MDS - ATP ATP LAMP MDS L. mocytogenes Listeria Ready-to-eat M MDS L monocytogenes Readyto-eat 図 1.MDS 法を用いた遺伝子増幅産物の検出原理

3 M MDS L MDS MDS 実験材料および方法 1. 供試菌および供試食材と接種菌数の確認 L. monocytogenes ATCC Trypticase Soy Broth TSB Difco. % Yeast extract TSBYE, Ready-to-eat L. monocytogenes Tripticase Soy Agar TSA; Difco. % Yeast extract TSAYE L. monocytogenes 2. 牛挽肉からのL. monocytogenes 検出感度試験 L. monocytogenes Fraser M Fraser Fraser ml g Fraser ml Fraser ml L. monocytogenes Fraser Fraser CFU/mL CFU/mL 3.Ready-to-eat 食品からの MDS による検出評価試験 L. moncytogenes CFU/mL. ml g ISO - MDS ISO - Demi-Fraser L Fraser L MDS MDS ISO - MDS 4. 低濃度接種区における検出感度試験と前培養培地間比較試験 g L. moncytogenes CFU/mL ml g ml ml TSBYE L. moncytogenes L. moncytogenes 表 1.L. monocytogenes の接種試験による MDS 法の食材適合試験結果. Demi-Fraser Fraser ISO- - MDS ISO- - MDS

4 図 2.MDS と培養法での L. monocytogenes 検出比較実験のスキーム g TSBYE Demi-Fraser ml ml - MDS L. moncytogenes TSBYE Demi-Fraser. ml ml Fraser L. moncytogenes ml Fraser 5. 培養法と抗体キットによるL. monocytogenes の検出と確認 L. monocytogenes ALOA L PBS L SiglePath L mono (Merck) L. monocytogenes L SiglePath L mono L. monocytogenes 6.MDS による核酸抽出法および遺伝子増幅反応による検出 MDS M Molecular Detection Assay Listeria monocytogenes M M Molecular Detection Instrument M L M Lysis tube Lysis tube - Lysis tube Lysis tube L M Molecular Detection Instrument / L. monocytogenes

5 結果 1. 牛挽肉における検出感度試験 Fraser Fraser L. monocytogenes CFU/mL L MDS CFU/mL Data not shown 2.Ready-to-eat 食品でのMDS 法の検出評価試験 CFU/ g L. moncytogenes MDS Demi-Fraser MDS. ml ml Fraser MDS 3. 低濃度接種区における検出感度試験と前培養培地間比較試験 L. monocytogenes. MPN/ g Demi-Fraser MDS MDS No. Fraser MDS No. MDS Fraser PCR L. monocytogenes Demi-Fraser Fraser TSBYE TSBYE MDS MDS MDS 表 2. 牛挽肉を用いた L. monocytogenes の培地間評価および検出試験結果. TSBYE Demi-Fraser Demi-Fraser Fraser SampleNo. h h SampleNo. h h ISO - MDS ISO - MDS ISO - MDS ISO - MDS

6 考察 L. monocytogenes ATP ATP L. monocytogenes MDS L L L CFU/ L. x CFU/mL copy CFU/mL MDS L. monocytogenes Demi-Fraser Fraser MDS L. monocytogenes TSBYE MDS Demi-Fraser Fraser MDS MDS MDS TSBYE MDS MDS TSBYE MDS MDS Demi-Fraser Fraser L. monocytogenes L. monocytogenes 要約 L. monocytogenes Ready to eat M Molecular detection system MDS. MPN/g g MDS ISO - MDS MDS L. monocytogenes

7 参考文献, -,,, ISO -, Microbiology of food and animal feeding stuffs -- Horizontal method for the detection and enumeration of Listeria monocytogenes. ( ). FDA: Bacteriological Analytical Manual, Capture ( ). Kawasaki, S., Horikoshi, N., Okada, Y., Takeshita, K., Sameshima, T. and Kawamoto, S., Multiplex realtime polymerase chain reaction assay for simultaneous detection and quantification of Salmonella species, Listeria monocytogenes, and Escherichia coli O157: H7 in ground pork samples. Foodborne Pathog. Dis., 7, - ( ). Li, F., Zhao, C., Zhang, W., Cui, S., Meng, J., Wu, J., and Zhang, D. Y., Use of ramification amplification assay for detection of Escherichia coli O :H and other E. coli Shiga toxin-producing strains. J. Clin. Microbiol., 43, -, ( ). Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T., Loopmediated isothermal amplification of DNA. Nucl. Acids Res., 28, E, ( ). Fortes, E. D., David, J., Koeritzer, B., Wiedmann, M., Validation of the M molecular detection system for the detection of listeria in meat, seafood, dairy, and retail environments. J. Food Prot., 76, -. ( ). Kawasaki, S., Horikoshi, N., Takeshita, K., Sameshima, T., and Kawamoto, S., Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O : H in meat samples. J. Food Prot. 68, -. ( ).

農研機構研究報告　食品研究部門　第1号

農研機構研究報告　食品研究部門　第1号 Bull. NARO. Food. ResNo., 研究ノート 3M TM 病原菌自動検出システム改良サルモネラ検出キット MDA2 SAL の評価 Fia Noviyanti - -- - -- - - - - Evaluation of 3M TM Molecular Detection System MDA2 SAL for the detection of Salmonella from food