한수지 47(5), 556-561, 2014 Original Article Kor J Fish Aquat Sci 47(5),556-561,2014 MPN 및 H-NS 유전자를표적으로하는 PCR assay 를병용한장염비브리오 (Vibrio parahaemolyticus) 의정량 김태옥 박권삼 * 군산대학교식품생명공학과 Quantification of Vibrio parahaemolyticus Using a Most Probable Number-Polymerase Chain Reaction Assay Targeting the H-NS gene Tae-Ok Kim and Kwon-Sam Park* Department of Food Science and Biotechnology, Kunsan National University, Gunsan 573-701, Korea We applied a combination of most probable number-polymerase chain reaction (MPN-PCR) methods using a PCR procedure targeting the H-NS (VP1133) gene to detect Vibrio parahaemolyticus presence and density in seawater as well as within short-necked clam Ruditapes philippinarum tissues collected from Gomso Bay, Korea. In 30 seawater samples, V. parahaemolyticus levels ranged from less than 1.8 to 1.1 10³ MPN/100 ml, and samples from August showed higher than those from other months. Furthermore, the levels of V. parahaemolyticus in six short-necked clam samples ranged from 7.8 10² to 2.1 10³ MPN/100 g, approximately 2.5 times higher than in seawater samples from the corresponding month. Our results provide data on V. parahaemolyticus contamination in seawater and shortnecked clam tissues, and help to improve quantitative methods of assessing V. parahaemolytcius levels. Key words: H-NS gene, Most probable number, Polymerase chain reaction, Vibrio parahaemolyticus 서론 (Vibrio parahaemolyticus),,, (Sakazaki et al., 1968; Blake et al., 1980; Honda and Iida, 1993). 97% (thermostable direct hemolysin, TDH) (TDH-related hemolysin, TRH) (Shirai et al., 1990; Honda and Iida, 1993). 2002 2013 12 16.5% 8.2% (MFDS, 2014)., 3,, 4 (MFDS, 2014). 17 10 (Kaneko and Colwell, 1975; Makino et al., 2003). 8-12 (3-4 ) (Makino et al., 2003). http://dx.doi.org/10.5657/kfas.2014.0556 Kor J Fish Aquat Sci 47(5) 556-561, October 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/)which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 26 September 2014; Revised 7 October 2014; Accepted 8 October 2014 *Corresponding author: Tel: +82. 63-469-1822 Fax: +82. 63-469-7448 E-mail address: parkks@kunsan.ac.kr Copyright 2014 The Korean Society of Fisheries and Aquatic Science 556 pissn:0374-8111, eissn:2287-8815
장염비브리오의정량 557 PCR assay, most probable number (MPN) (Copin et al., 2012), DNA hybridization (Wang et al., 2011), Chromogenic agar (Su et al., 2005) PCR assay (Kim et al., 1999; Kim et al., 2008; Yu et al., 2010). PCR assay tdh, trh, toxr, rpos, irgb gyrb (Venkateswaran et al., 1998; Bej et al., 1999; Kim et al., 1999; Yu et al., 2010). H-NS (histone-like nucleoid structuring) AT-rich DNA,, V. cholerae (Fang and Rimsky, 2008). 2003 RIMD2210633 DNA H-NS (VP1133) (Makino et al., 2003), DIG-labeling probe dot-blot hybridization 46 H-NS (Park et al., 2005). No et al. (2011) PCR assay H-NS H-NS. most probable number (MPN) H-NS 1km W-1 S-1 W-5 Buan-gun W-2 S-2 Fig. 1. Location of sample collection stations in Gomso Bay from June to November 2013., seawater sampling stations;, shortnecked clam sampling stations. S-3 Gochang-gun W-3 W-4 W N S E PCR assay. 시료채취지점및시료채취 재료및방법 Fig. 1. 2013 6 11 5 1 6, 3 2013 7 8 2. 250 ml,. 10., ph, (YSI556 multiprobe system; Yellow Springs, YSI Life Science, OH, USA). 균수조정 PCR assay 3% sodium chloride Luria-Bertani (1% tryptone, 0.5% yeast-extract, 3% NaCl) broth RIMD2210633 (12,000 rpm, 2 min). 1.0 ml PBS (phosphate buffered saline, ph 7.2) PBS. 10 µl counting chamber (Paul Marienfeld Gmbh&Co, KG Lauda-Konigshofen, Germany) (Olympus CX31RBSF, Olympus Optical Co., LTD. Tokyo, Japan) 10 PCR assay. 장염비브리오의정량시험 Recommended Procedures for the Sea Water and Shellfish (APHA, 1970). 2% sodium chloride alkaline peptone water (Merck, Darmstadt, Germany) 10 ml 3 10, 1, 0.1 0.01 ml 35 16. 25 g Waring Blender cup (Torrington, USA) 9 (phosphate buffered saline, ph7.4) 90 10 3 35 16. 1.0 ml eppendorf tube
558 김태옥ㆍ박권삼 (12,000 rpm, 2 min). 0.2 ml 98 5 H-NS PCR assay DNA. H-NS (most probable number, MPN) 100 ml 100 g. PCR assay 조건 EmeraldAmp GT PCR Master Mix (Takara, Japan). hns toxr 2.0 pmol, 50 µl. PCR 95 1 3 95 30, 55 30, 72 30 30 DNA. DNA 1.5% agarose gel ethidium bromide Vilber Lourmat (Bio-Paint ST4, Marne-la-Vallee, France) Gel-Doc system. 결과및고찰 PCR assay 에의한장염비브리오의검출최소농도의검토 PCR assay. DNA groel, PCR assay 100 pg (Hossain et al., 2011), H-NS PCR assay 0.14 pg (No et al., 2011), O-serogroup PCR assay 1.0 ng (Chen et al., 2012). PCR assay 10 colony forming unit (CFU) (Wei et al., 2014). H-NS PCR assay 0.8 10 CFU 0.8 10 0 CFU. Fig. 2 H-NS 0.8 10 0 CFU DNA 0.8 10 CFU 0.8 10¹ CFU 465 bp DNA. 0.8 10¹ CFU DNA. toxr PCR assay H-NS ( ). PCR assay 8 CFU (Wei et al., 2014). 8 CFU. 해수중의장염비브리오정량 H-NS primer set PCR assay 2013 6 11 6 30.,, ph Table 2. 6 21-23 8 30 11 15-16. ph, 8.,, ph M 1 2 3 4 5 465 bp Fig. 2. Agarose gel electrophoresis of DNA products amplified in PCR assay using hns primers. M, 100 bp ladder marker (Takara, Japan); lane 1, 0.8 10⁴CFU; lane 2, 0.8 10³ CFU; lane 3, 0.8 10² CFU; lane 4, 0.8 10¹ CFU; lane 5, 0.8 10 0 CFU. Table 1. Oligonucleotide primers used in this study Gene Oligonucleotide sequence Amplicon size (bp) Reference toxr (VP0820) 5'-AGCCCGCTTTCTTCAGACTC-3' 5'-AACGAGTCTTCTGCATGGTG-3' 399 Kim et al., 1999 hns (VP1133) 5'-AAACACGTTAACCTATTAATAGG-3' 5'-AACGGGAGCCTTTTTAAACAAGA-3' 465 No et al., 2011
장염비브리오의정량 559. 21-23 6 20-78 MPN/100 ml ( 48.4 MPN/100 ml), 7 200-780 MPN/100 ml ( 472 MPN/100 ml), 8 780-1,100 MPN/100 ml ( 972 MPN/100 ml), 9 110-170 MPN/100 ml ( 134 MPN/100 ml), 10 11-46 MPN/100 ml ( 27 MPN/100 ml), 11 <1.8-2.0 MPN/100 ml ( 1.9 MPN/100 ml). 8. DePaola et al. (1990) 17 CFU/100 ml,. Cantet et al. (2013) 1.0 >1.1 10²MPN/100 ml 10. Table 2, ph Table 2. Levels of Vibrio parahaemolytcius in surface seawater in Gomso Bay from June to November 2013 Month June July Aug. Sep. Oct. Nov. St. Temp. ( ) Sal. (ppm) ph DO (mg/l) V. parahaemolyticus (MPN/100 ml) W-1 21.85 30.23 8.08 7.55 78 W-2 21.29 30.55 8.13 8.14 46 W-3 21.20 30.54 8.12 8.34 20 W-4 23.31 29.71 8.04 7.56 20 W-5 22.22 30.15 8.08 7.86 78 W-1 26.87 29.29 7.73 6.53 460 W-2 24.99 29.48 7.72 6.06 780 W-3 25.06 29.06 7.75 6.52 200 W-4 25.94 29.42 7.73 6.54 460 W-5 26.75 29.72 7.68 6.52 460 W-1 30.28 30.71 7.88 5.84 780 W-2 29.24 30.84 7.86 5.52 1,100 W-3 29.48 30.46 7.88 5.57 780 W-4 30.11 30.28 7.87 6.38 1,100 W-5 29.51 30.20 7.86 5.18 1,100 W-1 22.91 30.19 7.72 6.16 110 W-2 22.98 30.34 7.73 6.62 110 W-3 23.15 30.22 7.74 6.69 170 W-4 24.18 30.85 7.73 6.23 140 W-5 23.27 30.61 7.78 6.18 140 W-1 20.52 29.60 7.89 6.76 46 W-2 20.38 29.55 7.96 7.35 11 W-3 20.30 29.56 7.94 7.31 46 W-4 20.84 29.50 7.85 6.94 20 W-5 20.54 29.27 7.90 7.27 11 W-1 15.97 30.52 8.09 6.03 <1.8 W-2 16.22 30.56 7.99 6.18 2.0 W-3 16.14 30.64 8.03 6.14 2.0 W-4 15.19 30.15 8.07 6.03 <1.8 W-5 15.63 30.43 8.07 6.01 <1.8 Total 15.19~30.28 29.06~30.85 7.68~8.13 5.18~8.34 <1.8~1,100 St., Station; Temp., Temperature; Sal., Salinity; DO., Dissolved oxygen.
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