한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 MPN 및 H-NS 유전자를표적으로하는 PCR assay 를병용한장염비브리오 (Vibrio parahaemolyticus)

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1 한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,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 , 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 ³ 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 ² to ³ 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) % 8.2% (MFDS, 2014)., 3,, 4 (MFDS, 2014) (Kaneko and Colwell, 1975; Makino et al., 2003) (3-4 ) (Makino et al., 2003). Kor J Fish Aquat Sci 47(5) , October 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( 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: Fax: address: parkks@kunsan.ac.kr Copyright 2014 The Korean Society of Fisheries and Aquatic Science 556 pissn: , eissn:

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.

2 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) RIMD 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 , 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 RIMD (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, ml g Waring Blender cup (Torrington, USA) 9 (phosphate buffered saline, ph7.4) 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.

3 558 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 , 55 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 CFU CFU. Fig. 2 H-NS CFU DNA CFU ¹ CFU 465 bp DNA ¹ CFU DNA. toxr PCR assay H-NS ( ). PCR assay 8 CFU (Wei et al., 2014). 8 CFU. 해수중의장염비브리오정량 H-NS primer set PCR assay ,, ph Table ph, 8.,, ph M 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, ⁴CFU; lane 2, ³ CFU; lane 3, ² CFU; lane 4, ¹ CFU; lane 5, 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

4 장염비브리오의정량 MPN/100 ml ( 48.4 MPN/100 ml), MPN/100 ml ( 472 MPN/100 ml), ,100 MPN/100 ml ( 972 MPN/100 ml), MPN/100 ml ( 134 MPN/100 ml), MPN/100 ml ( 27 MPN/100 ml), 11 < 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 W W W W W W W W W W W ,100 W W ,100 W ,100 W W W W W W W W W W W <1.8 W W W <1.8 W <1.8 Total 15.19~ ~ ~ ~8.34 <1.8~1,100 St., Station; Temp., Temperature; Sal., Salinity; DO., Dissolved oxygen.

5 560 김태옥ㆍ박권삼 (Kaneko and Colwell, 1975). 바지락중의장염비브리오정량 Fig. 1 6 MPN-PCR assay. 7, 780-1,700 MPN/100 g ( 1,193 MPN/100 g), 8 1,700-2,100 MPN/100 g ( 1,967 MPN/100 g) , 2.5, DePaola et al. (1990) 100. Table 3. Levels of Vibrio parahaemolytcius in short-necked clam Ruditapes philippinarum in Gomso Bay from July to August 2013 Month July Aug. St., Station. St. V. parahaemolyticus (MPN/100 g) S-1 1,700 S-2 1,100 S S-1 2,100 S-2 2,100 S-3 1,700. 사사 2013 ( ) (2013R1A1A ). References A.P.H.A Recommended procedures for the examination of seawater and shellfish. 4th Ed. American Public Health Association, Washington DC, U.S.A, Bej AK, Patterson DP, Brasher CW, Vickery MC, Jones DD and Kaysner CA Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. J Microbiol Meth 36, Blake PA, Weaver RE and Hollis DG Diseases of humans (other than cholera) caused by vibrios. Annu Rev Microbiol 34, Cantet F, Hervio-Heath D, Caro A, Le Mennec C, Monteil C, Quemere C, Jolivet-Gougeon A, Colwell RR and Monfort P Quantification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons. Res Microbiol 164, org/ /j.resmic Chen M, Guo D, Wong HC, Zhang X, Liu F, Chen H, Chen M, Liu B, Wang L, Wu F and Feng L Development of O- serogroup specific PCR assay for detection and identification of Vibrio parahaemolyticus. Int J Food Microbiol 159, Copin S, Robert-Pillot A, Malle P, Quilici ML and Gay M Evaluation of most-probable-number-pcr method with internal amplification control for the counting of total and pathogenic Vibrio parahaemolyticus in frozen shrimps. J Food Prot 75, DePaola A, Hopkins LH, Peeler JT, Wentz B and McPhearson RM Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters. Appl Environ Microbiol 56, Fang FC and Rimsky S New insights into transcriptional regulation by H-NS. Curr Opin Microbiol 11, Honda T and Iida T The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysins. Rev Med Microbiol 4, Hossain MT, Kim EY, Kim YR, Kim DG and Kong IS Application of groel gene for the species-specific detec-

6 장염비브리오의정량 561 tion of Vibrio parahaemolyticus by PCR. Lett Appl Microbiol 54, x. Kaneko T and Colwell RR Adsorption of Vibrio parahaemolyticus onto chitin and copepods. Appl Microbiol 29, Kim YB, Okuda J, Matsumoto C, Takahashi N, Hashimoto S and Nishibuchi M Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxr gene. J Clin Microbiol 37, Makino K, Oshima K, Kurokawa K, et al., Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V. cholerae. Lancet 361, Ministry of Food and Drug Safety (MFDS) Korea food code. Retrieved from do?nmenucode=20 on September 15. No AR, Okada K, Kogure K and Park KS Rapid detection of Vibrio parahaemolyticus by PCR targeted to the histone-like nucleoid structure (H-NS) gene and its genetic characterization. Lett Appl Microbiol 53, dx.doi.org/ /j x x. Park KS, Arita M, Iida T and Honda T vpah, a gene encoding a novel Histone-like nucleoid structure-like protein that was possibly horizontally acquired, regulates the biogenesis of lateral flagella in trh-positive Vibrio parahaemolyticus TH3996. Infect Immun 73, Sakazaki R, Tamura K, Kato T, Obara Y and Yamai S Studies on the enteropathogenic, facultatively halophilic bacterium, Vibrio parahaemolyticus. 3. Enteropathogenicity. Jpn J Med Sci Biol 21, Shirai H, Ito H, Hirayama T, Nakamoto Y, Nakabayashi N, Kumagai K, Takeda Y and Nishibuchi M Molecular epidemiological evidence for association of thermostable direct hemolysin (TDH) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis. Infect Immun 58, Su YC, Duan J and Wu WH Selectivity and specificity of a chromogenic medium for detecting Vibrio parahaemolyticus. J Food Prot 68, Venkateswaran K, Dohmoto N and Harayama S Cloning and nucleotide sequence of the gyrb gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp. Appl Environ Microbiol 64, Wang R, Huang J, Zhang W, Lin G, Lian J, Jiang L, Lin H, Wang S and Wang S Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA-DNA hybridization on a microarray. J Genet Genomics 38, Wei S, Zhao H, Xian Y, Hussain MA and Wu X Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control. Diagn Microbiol Infect Dis 79, Yu S, Chen W, Wang D, He X, Zhu X and Shi X Speciesspecific PCR detection of the food-borne pathogen Vibrio parahaemolyticus using the irgb gene identified by comparative genomic analysis. FEMS Microbiol Lett 307,

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