한수지 50(6), 669-674, 2017 Original Article Korean J Fish Aquat Sci 50(6),669-674,2017 마비성패류독소검출을위한분석법비교 이가정 * 권순재 정연중 손광태 하광수 1 목종수 1 김지회 2 국립수산과학원남동해수산연구소, 1 국립수산과학원식품위생가공과, 2 국립수산과학원연구기획과 Comparison of Analytical Methods for the Detection of Paralytic Shellfish Toxins (PSTs) Ka Jeong Lee*, Soon Jae Kwon, Yeoun Joong Jung, Kwang Tae Son, Kwang Soo Ha 1, Jong Soo Mok 1 and Ji Hoe Kim 2 Southeast Sea Fisheries Research Institute, National Institute of Fisheries Science, Tongyeong 53085, Korea 1 Food Safety and Processing Research Division, National Institute of Fisheries Science, Busan 46083, Korea 2 Research and Development Planning Division, National Institute of Fisheries Science, Busan 46083, Korea Paralytic shellfish toxins (PSTs) are produced by marine dinoflagellate phytoplankton Alexandrium spp. and Gymnodinium spp. These toxins accumulate in filter feeding organisms such as bivalves and the ingestion of contaminated shellfish can cause illness in humans. The mouse bioassay (MBA) has been the preferred PST testing method worldwide for more than 50 years. However, this assay has several disadvantages, such as detection limits, non-toxicprofiles, and the ethical issues of using animals. The aim of this study was to establish an alternative to the MBA method for testing for PSTs. We optimized the analysis conditions of a post-column oxidation-high performance liquid chromatography (PCOX-HPLC) method and the Scotia Rapid Test Kit, and then compared the accuracy of these methods to the MBA method. The results demonstrated a strong correlation between the PCOX-HPLC method and the MBA, although the PCOX-HPLC method required expensive equipment and standard material, and was time consuming. The Scotia Rapid Test Kit promises to be a useful tool, as it provided rapid and qualitative results, although the method sometimes gave a false positive result that could not be explained by toxin profiles. Key words: Paralytic shellfish toxins (PSTs), Mouse bioassay (MBA), Scotia Rapid Test Kit, Post-column oxidation- HPLC (PCOX-HPLC) 서론 (Paralytic Shellfish Toxins, PSTs) Alexandrium spp., Gymnodinium spp. (filter feeding) (Thomas et al., 2006).,, (NIFS, 2016) (Fig. 1).,. AOAC 959.08 official method (OM) (mouse bioassay; AOAC MBA) (AOAC, 2005), 40 µg/100 g STX equivalent (equiv.) 50 (Schantz et al., 1958)., (Hess et al., 2006), (Park et al., 1986).,. AOAC (Association of analytical communities) HPLC https://doi.org/10.5657/kfas.2017.0669 Korean J Fish Aquat Sci 50(6) 669-674, December 2017 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 31 October 2017; Revised 16 November 2017; Accepted 8 December 2017 *Corresponding author: Tel: +82. 55. 640. 4761 Fax: +82. 55. 641. 2036 E-mail address: kajlee@korea.kr Copyright 2017 The Korean Society of Fisheries and Aquatic Science 669 pissn:0374-8111, eissn:2287-8815
670 이가정ㆍ권순재ㆍ정연중ㆍ손광태ㆍ하광수ㆍ목종수ㆍ김지회 pre-column oxidation-hplc postcolumn oxidation-hplc (PCOX-HPLC) (AOAC 2011.02 Official method) (AOAC, 2011)., (US National shellfish sanitation program, NSSP) PCOX-HPLC Scotia rapid test (NSSP, 2005)., (Mytilus galloprovincialis), (Crassostrea gigas), (Ruditapes philippinarum) (Styela clava), PCOX-HPLC (Scotia Rapid Test Kit). 재료및방법 시료채취및운반 (Mytilus galloprovincialis), (Crassostrea gigas), (Ruditapes philippinarum) (Styela clava), 2015 3-5., 8.7-17.4, 31.2-33.7 psu. 시약및표준물질 (analytical grade, Merck, Darmstadt, Germany) 0.1 N,, PCOX-HPLC. PCOX-HPLC Song et al. (2013), NEO (neosaxitoxin), STX (saxitoxin), dcstx (decarbamoylsaxitoxin), GTX1 (gonyautoxin-1), GTX2 (gonyautoxin-2), GTX3 (gonyautoxin-3), GTX4 (gonyautoxin-4), GTX5 (gonyautoxin-5), dcgtx2 (decarbamoylgonyautoxin-2), dcgtx3 (decarbamoylgonyautoxin-3), C1 (N-sulfocarbamoylgonyautoxin-C1) C2 (N-sulfocarbamoylgonyautoxin-C2) NRC (Halifax, Canada)., Scotia rapid testing Ltd. (Halifax, Canada). 마우스시험 (KMFDS, 2015) 1 ml 19-21 g ICR Sommer s Table. No. of analytes (n) 2000 1500 1000 500 0 Fig. 1. Number of analytes for paralytic shellfish toxins (2006-2015). PCOX-HPLC 시험 PCOX-HPLC AOAC (2011) Song et al. (2013),., HPLC fluorescence detector e2695 2475 FLR Detector (Waters, MA, USA), detector excitation wavelength 330 nm, emission wavelength 390 nm. Post-column reaction system postcolumn derivatizer (Waters, MA, USA) 80. GTXs/STXs column 40 100% A 0.8 ml/ min, C1 C2 column 15 100% C 0.8 ml/min. column GTX STX ZorbaxBonus-RP (Agilent, CA, USA, 863668-901T, 4.6 150 mm, 3.5 µm), C1 C2 Phenosphere-NEXT, (Phenomenex, CA, USA, 00G-4307-E0,4.6 250 mm, 5 µm). single point calibration peak saxitoxin µg/100 g STX equiv.. 면역시험키트시험 Scotia buffer positive ( ) negative ( ). 결과및고찰 마우스시험법 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 Year 4
마비성패류독소분석법비교 671 PCOX-HPLC. 52 10, 6 7,. 7 85-627 µg/100 g STX equiv.., (Lee et al., 2006). 6 42-57 µg/100 g STX equiv., 7 42-94 µg/100 g STX equiv.. (filter feeding) (Kim et al., 1998),. PCOX-HPLC 법 Post column oxidation HPLC (PCOX-HPLC). 52,,, GTX2,3 45-92%, GTX1,4 1-48%, STX 18-39%, (Table 1)., C1 C2. PCOX-HPLC (r 2 ) 0.9878 (Fig. 3). 40 µg/100 g STX equiv. PCOX- HPLC. PCOX-HPLC,, (Rourke et al., 2008).,. 면역시험키트법, PCOX-HPLC. Scotia rapid test Jellet rapid test. polyclonal PSP toxin antibodies (lateral flow immunochromatography) (A) (B) Fig. 2. Results of Scotia rapid test strips. C, control line; T, test line; A, Negative (not detected); B, Positive(detected). MBA (μg/100 g STX equiv.) 700 600 500 400 300 200 100 0 0 100 200 y = 0.9454x + 4.4877 R 2 = 0.9878 MBA HPLC 300 400 500 600 700 PCOX-HPLC (μg/100 g STX equiv.) Fig. 3. Correlation between mouse bioassay and post-column oxidation-high performance liquid chromatography (HPLC) method.. 35 40 µg/100 g STX equiv.., running buffer 35. test line ( T line) control line ( C ) (, positive) (, negative). Fig. 2, test line, (Cefas, 2007). table 1.,, false positive ( ) false negative ( ).,
672 이가정ㆍ권순재ㆍ정연중ㆍ손광태ㆍ하광수ㆍ목종수ㆍ김지회 Table 1. Comparison of MBA, PCOX-HPLC methods and Scotia rapid kit for the detection of paralytic shellfish toxins Sample Mussel Oyster Short neck Clam PSTs Toxicity by MBA 1 (µg/100 g STX equiv.) Scotia Rapid Kit (µg/100 g STX equiv.) PSTs concentration by PCOX-HPLC % Molar fraction Total STX 3 dc-stx 4 NEO 5 GTX1,4 6 GTX2,3 7 GTX5 8 dc-gtx2,3 9 1 ND 2-10 0 0 0 0 0 0 0 0 2 ND - 25 27 1 1 2 67 1 1 3 85 + 11 82.5 24 2 1 2 69 1 1 4 627 + 597 25 2 1 3 66 1 2 5 213 + 234 18 2 1 3 74 1 1 6 397 + 325 26 1 1 1 70 0 1 7 379 + 394 25 2 1 2 70 0 1 8 296 + 304 25 1 1 1 70 0 1 9 163 + 142 33 1 1 2 63 0 0 10 73 + 67 30 1 1 1 63 2 2 11 56 + 52 28 1 1 2 68 0 1 12 43 + 41 28 2 1 2 67 0 1 13 ND - 16 39 1 1 2 54 1 3 1 ND - 0 0 0 0 0 0 0 0 2 ND - 0 0 0 0 0 0 0 0 3 ND - 18 0 1 0 39 51 9 0 4 57 + 49 0 6 0 42 45 4 3 5 42-39.1 0 1 0 42 55 2 0 6 45-43.3 0 4 0 35 53 3 5 7 42 + 41 0 0 0 9 91 0 0 8 51 + 54 0 1 0 12 84 1 0 9 42 + 39 0 1 0 7 92 0 0 10 ND - 22 0 2 0 35 51 10 2 11 ND - 12 0 0 0 48 34 15 3 12 ND - 0 0 0 0 0 0 0 0 13 ND - 0 0 0 0 0 0 0 0 1 ND - 0 0 0 0 0 0 0 0 2 ND - 0 0 0 0 0 0 0 0 3 42 + 41 25 1 0 2 71 0 1 4 53 + 57 32 1 1 1 62 0 1 5 49 + 42 32 1 1 1 63 0 1 6 42 + 40 27 1 1 1 70 1 1 7 42 + 45 18 4 1 4 69 2 2 8 94 + 81 30 1 0 1 66 0 1 9 59 + 66 32 1 1 1 64 0 1 10 ND - 28 35 1 1 1 60 0 2 11 ND - 0 0 0 0 0 0 0 0 12 ND - 0 0 0 0 0 0 0 0 13 ND - 0 0 0 0 0 0 0 0
마비성패류독소분석법비교 673 Table 1. Continued Sample Warty Sea Squirt PSTs Toxicity by MBA 1 (µg/100 g STX equiv.) Scotia Rapid Kit (µg/100 g STX equiv.) PSTs concentration by PCOX-HPLC % Molar fraction Total STX 3 dc-stx 4 NEO 5 GTX1,4 6 GTX2,3 7 GTX5 8 dc-gtx2,3 9 1 ND - 0 0 0 0 0 0 0 0 2 ND - 0 0 0 0 0 0 0 0 3 ND - 0 0 0 0 0 0 0 0 4 ND - 0 0 0 0 0 0 0 0 5 ND - 0 0 0 0 0 0 0 0 6 ND - 0 0 0 0 0 0 0 0 7 ND - 0 0 0 0 0 0 0 0 8 ND - 0 0 0 0 0 0 0 0 9 ND - 0 0 0 0 0 0 0 0 10 ND - 0 0 0 0 0 0 0 0 11 ND - 0 0 0 0 0 0 0 0 12 ND - 0 0 0 0 0 0 0 0 13 ND - 0 0 0 0 0 0 0 0 1 MBA, Mouse Bioassay; 2 ND, Not detected; 3 STX, Saxitoxin; 4 dc-stx, Decarbamoylsaxitoxin; 5 NEO, Neosaxitoxin; 6 GTX1,4, Gonyautoxin 1,4; 7 GTX2,3, Gonyautoxin 2,3; 8 GTX5, Gonyautoxin 5; 9 dc-gtx2,3, decarbamoylgonyautoxin 2,3; 10 -, negative (not detected); 11 +, positive (detected). false positive, 42-45 µg/100 g STX equiv. 2 false negative., false positive false negative. false positive false negative negative. STX, GTX2,3, C1,2, GTX5 dcstx, NEO GTX1,4 (Laycock et al., 2001), STX (100%), dcstx (30%), GTX2,3 (60%), GTX5 (40%), C1,2 (33%), dcgtx2,3 (7%), NEO (20%) GTX1,4 (2%) (Jellet et al., 2002)., GTX2, GTX3 23.3 µg/100 g STX equiv., STX 19.9 µg/100 g STX equiv., GTX1,4 345.2 µg/100 g STX equiv. (Cefas, 2007). 2 false negative GTX2,3 (53-55%) 23.3 µg/100 g STX equiv.., positive negative. 분석방법의비교,, PCOX-HPLC. PCOX-HPLC, PCOX- HPLC,,.,, 80 µg/100 g STX equiv., positive negative., false positive, 40 µg/100 g STX equiv. false positive (Cost et al., 2009; Oshiro et al., 2009).,, PCOX-HPLC (FAO/WHO, 2016)., PCOX-HPLC,.,.
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