3 5 Vol. 3 No. 5 2012 10 Journal of Food Safety and Quality Oct., 2012 刘莹 *, 陈溪, 崔晗, 黄大亮, 张晓林 (, 116400) 摘要 : 目的 (MBA)(ELISA), 方法 MBA ELISA (DSP) (PSP), ELISA (ASP) (NSP) 结果 2009~2011 8 67, : DSP PSP, ELISA ASP NSP, ASP NSP, 结论 关键词 : ; ; ; ; Comparison of two screening methods in detection of shellfish poisons LIU Ying *, CHEN Xi, CHUI Han, HUANG Da-Liang, ZHANG Xiao-Lin (Dalian Entry-Exit Inspection and Quarantine Bureau Offices in Zhuanghe, Zhuanghe 116400, China) ABSTRACT: Objective To determine four kinds of toxins in shellfish by using the methods of mouse bioassay(mba) and enzyme-linked immunosorbent assay(elisa) and provide references for the rapid detection of toxins in shellfish. Methods Diarrhetic shellfish poison and paralytic shellfish poison were detected by MBA and ELISA, while amnesic shellfish poison and neurotoxic shellfish poison were detected by ELISA. Results The experiment was conducted using a total of 8 kinds of 67 samples and the results showed that there were no differences between the detection results of diarrhetic shellfish poison and paralytic shellfish poison using the two methods. The result obtained by MBA was in good accordance with that of ELISA. Conclusion These two kinds of rapid screening methods in the detection of shellfish poison has its own application scope. The laboratory can choose a suitable detection method based on different situations. KEY WORDS: shellfish poison; mouse bioassay; enzyme-linked immunosorbent assay(elisa); rapid screening; detection 4 : (diarrhetic shellfish poison, DSP) (paralytic shellfish poison, PSP) (amnesic shellfish poison, ASP) (neurotoxic shellfish poison, NSP)DSP, : ( OA DTX) ( PTX) ( YTX) PSP,, : (carbamoyl toxin) N (N-su1fo carbamoyl toxin) (decarbamoyl toxin) (saxitoxins, STX) (neosaxitoxins, neo- STX)(gonyautoxins, GTX1-4) -N- GTX5 GTX6 dcstx dcneostx dcgtx1-4 ASP (domoic acid, DA), 7 NSP * 通讯作者 :,,, : E-mail: lnciqly@126.com
496 3, 13, BTX-A, B B( hemibrevetoxins B) [1] 2002 /225 /EC DSP : OA/DTX PTX 160 μg OA eq/kg; YTX 1 mg YTX eq/kg; AZA 160 μg AZA eq/kg 100 mg 80 g ( STX ), 4 MU/ g ASP 20 mg DA/kg, NSP 0.8mg/kg ( : 20 MU/100 g) [2] (mouse bioassay, MBA) (ELISA), 1 材料与仪器 1.1 材料与试剂 (okadaic acid, OA) (saxitoxin, STX) (domoic acid, DA) (National Research Council of Canada, NRC); : Abraxis ; : 18.0~21.0 g ICR ( ); ; 1%-60 ( );, 3 ~4 1.2 仪器与设备 ( Sunrise ); T-18 ( IKA ); R-215 ( BUCHI ); ( Eppendorf ); MS3 ( IKA ); ( ); L15/12 ( LAC ); 3K15 ( SIGMA ); : 1 ml; PL203 ( ) 2 实验部分 2.1 试样的制备,,,,,, 500 g 3 10 5 min( ),, 2.2 小鼠生物检测法 [3] 2.2.1 DSP 小鼠生物检测法 : 200 g ( 0.1 g),,, 3 2 min;, ;,, 56 ±1, 100~200 ml,,, 2,, 50 ml, 1 % 60 10 ml : 18.0~20.0 g ICR 6,,, ; 1 ml 1% 60, 24 h, 2 3, 1 : 24 h,, 1, DSP : <0.05 MU/g, 2 3 1 [4, 5] 2.2.2 PSP 小鼠生物检测法 PSP : ph 3 l μg/ml, 3 ~4 10 15 20 25 30 ml 1 μg/ml 10 ml,
5, : 497 表 1 注射量与毒力的关系 Table 1 Relationship of injection dose and virulence (ml) 1) (g) (MU/g) 1.0 20 0.05 0.5 10 0.1 4 1.0 5 0.2 4 0.5 2.5 0.4 16 1.0 1.25 0.8 16 0.5 0.625 1.6 : 1), : 2.1 100 g 800 ml, 0.18 mol/l 100 ml,, ph 2.0~4.0 ;, 5 mol/l 0.1 mol/l ph,,,, 5 min,, 200 ml, ph 2.0~4.0(pH >4.5),,,, 3000 r/min 5 min, : 1), 10, 5~7 min, 5~7 min 1 18 ml +1 μg/ml 10 ml; 2 19 ml +1 μg/ml 10 ml; 3 20 ml +1 μg/ml 10 ml : C CF = (1) CMU : CF ; C STX, (μg/ml); CMU ( ) 10 CF, 表 2 石房蛤毒素标准液注射所得毒素转换系数 Table 2 Shellfish poison conversion coefficient of injected STX stranded solvent W t CF 1 19.86±0.16 354.00±34.79 0.220±0.02 2 19.96±0.22 347.50±42.38 0.207±0.03 3 19.98±0.18 344.50±41.26 0.199±0.02 : W (g); t (s) CF1=0.220 CF2=0.207 CF3=0.199 CF 0.209 2) 19.0~21.0 g ICR 6, (0.18 mol/l ), 1 ml, ( ), 1 2 7 min, 5 min,, (3 ), 5~7 min ;, 0.18 mol/l, ph 2.0~ 4.0 : PSP : 100 g PSP (2) X = CMU1 CF DF 200 (2) : X 100 g PSP (µg /100 g); CMU1 ; CF ; DF ; 200, (ml) 2.3 酶联免疫吸附法 2.3.1 ELISA 法毒素的提取 DSP : 80%(:, 80: 20); 1.3.1, ; 1 g ( 0.1 g) 6 ml 80%; 3500 r/min 10 min, ; 2 ml 80% 4 3500 r/min 10 min,, ; 80% 10 ml
498 3 PSP : 10 g 1.3.1 ( 0.1 g) 10 ml 80% 0.1 mol/l HCl, 5 min, ; 3500 r/min 10 min; 5 mol/l HCl ph 4.0, 10 ml ASP : 0.5 g 1.3.1 ( 0.01 g), 2 ml / (50/50), 1 min 3000 r/min 15 min, 0.45 μm, 20 ul 1 ml NSP : 1.00 g 1.3.1 ( 0.01 g), 40 ml, 9.0 ml /(9: 1, v/v);, 2 min; 3000 r/min 10 min,,,, 2.3.2 ELISA 法毒素的检测及计算 : ; ( ), 450 nm (OD), 15 min :,, (ng/ml),, DSP PSP ASP NSP 3-6 DSP PSP ASP NSP, DSP 10 μg/kg; PSP 20 μg/kg; ASP 1 μg/g; NSP 50 μg/kg Table 3 表 3 DSP 标准溶液吸光度值 Absorbance value of DSP stranded solvent (ng/ml) DSP 1 2 A 0.0 1.402 1.387 1.395 1.000000 B 0.1 1.109 1.110 1.110 0.795626 C 0.2 0.962 0.958 0.960 0.688419 D 0.5 0.691 0.694 0.693 0.496594 E 1.0 0.514 0.520 0.517 0.370742 F 2.0 0.362 0.358 0.360 0.258157 G 5.0 0.203 0.206 0.205 0.146648 Table 4 表 4 PSP 标准溶液吸光度值 Absorbance value of PSP stranded solvent (ng/ml) PSP 1 2 A 0.00 2.018 2.101 2.060 1.000000 B 0.02 1.790 1.788 1.789 0.868657 C 0.05 1.404 1.388 1.396 0.677834 D 0.10 0.927 0.929 0.928 0.450595 E 0.20 0.583 0.559 0.571 0.277252 F 0.40 0.364 0.314 0.339 0.164603
5, : 499 表 5 ASP 标准溶液吸光度值 Table 5 Absorbance value of ASP stranded solvent (ng/ml) NSP 1 2 A 0.00 0.901 0.868 0.885 1.000000 B 0.50 0.645 0.640 0.643 0.726399 C 1.00 0.518 0.513 0.516 0.582815 D 2.00 0.432 0.399 0.416 0.469757 E 5.00 0.231 0.209 0.220 0.248728 F 10.00 0.141 0.155 0.148 0.167326 表 6 NSP 标准溶液吸光度值 Table 6 Absorbance value of NSP stranded solvent (ng/ml) NSP 1 2 A 0.00 1.398 1.390 1.394 1.000000 B 0.01 0.927 0.933 0.930 0.667145 C 0.025 0.748 0.745 0.747 0.535509 D 0.05 0.692 0.641 0.667 0.478121 E 0.10 0.585 0.566 0.576 0.412841 F 0.25 0.483 0.452 0.468 0.335366 G 0.50 0.385 0.362 0.374 0.267934 H 2.00 0.151 0.149 0.150 0.107604 3 结果与讨论 3.1 结果 DSP PSP, DSP PSP ASP NSP, 7, PSP μg/kg, 2009~2011 8 67, DSP PSP, DSP PSP, ( 8) ELISA ASP NSP, ASP NSP, 3.2 讨论 3.2.1 两种快速筛选方法比较 DSP PSP NSP, ; ;,, ;,, ;, 3R ;,, ; ; [10] ; ELISA() RIA() EIA() S-PIA ( ) [11], ELISA (RIA),,,,,,,,, (AOAC) ELISA ELISA
500 3 1 2 3 4 5 6 7 8 (7 ) (8 ) (3 ) (6 ) (7 ) (11 ) (4 ) (8 ) Table 7 表 7 MBA 法和 ELISA 法测得样品中四种贝类毒素结果 Detection of 4 kinds of shellfish poisons with MBA and ELISA method MBA (MU/g) DSP PSP ASP NSP ELISA (μg/kg) MBA (μg /100g) ELISA (μg/kg) ASP ELISA (μg/ g) NSP ELISA (μg/ kg) ND 2 ND 75.24 810.5 ND ND 0.1 386 78.24 824.2 ND ND ND ND ND ND 1.24 ND ND ND ND ND ND 0.045 ND ND 61.86 685.8 ND ND 0.05 203 ND ND ND ND ND ND ND ND ND 0.061 ND ND ND ND 2.48 ND : 1 ASP NSP ; 2 ; 3 ND Table 8 表 8 实际应用中 ELISA 与 MBA 法检测四种贝类毒素结果 The detection results of 4 kinds of shellfish poisons with the MBA and ELISA method ELISA DSP ELISA PSP ELISA ASP NSP ASP 9 6 3 100% 6 3 100% 1 2 6 6 0 100% 6 0 100% 0 0 9 8 1 100% 8 1 100% 0 1 8 7 1 100% 6 2 100% 0 0 7 6 1 100% 6 1 100% 1 1 6 6 0 100% 6 0 100% 0 0 5 5 0 100% 5 0 100% 0 0 17 17 0 100% 17 0 100% 3 1 67 61 6 100% 60 7 100% 5 5 NSP,,,, 3.2.2 其他检测方法 (HPLC) HPLC,, (LC-UV) DA ; (LC-FL) OA STX dc-stx ( LC-MS) [12] HPLC : ; ;, ;, ;,, HPLC, HPLC [13]
5, : 501,,,, -,, (),, LC-MS(MS/MS), C 18, CN,, ( ), μg/kg,,, [14-16], 4 结论 DSP PSP ASP NSP : DSP PSP, DSP PSP ELISA ASP NSP, ASP NSP,,, 参考文献 [4] GB/T 5009.213-2008, [S]. [5] SC/T 3023-2004, [S]. [6] Association of Official Analytical Chemists (AOAC) (1984). Arlington, V.A. Official Methods of Analysis 18.086-18.092 (Adams, W.N. and Miescier, J. J.) J Assoc Off Anal Chem, 1980, 63: 1336. [7],,,. [J]., 2011, 39(26): 16070 16073, 16136. [8] SNT 1573-2005, [S]. [9] APHA, Method for Ptychodiscus brevis toxins in Laboratory procedures for the examination of seawater and shellfish, 5th ed.; American Public Health Association: Washington, DC, USA, 1985: 64 80. [10] John J Sullivan. (DSP) (PSP) [J], 1991, 10(4): 43. [11] Hallegraeff GM. Manual on Harmful Marine Microalgae. UNESCO, France: IOC Manuals and Guides No. 33. 1995: 182 199. [12] Fux E, McMillan D, Bire R, et al. Development of an ultraperformance liquid chromatography-mass spectrometry method for the detection of lipophilic marine toxins[j]. J Chromatogr A, 2007, 1157(1 2) :273 280. [13],. [J]., 2001, 12(2): 48. [14],. [J]., 2004, 14(1): 53 36. [15],,,. [J]., 2000, 24(9): 19 22. [16],,. [J]., 2000, 19(1): 73 78. ( 责任编辑 : 张宏梁 ) [1] Alexander J, Benford D, Boobis A, et al. Marine biotoxins in shellfish domoic acid[j]. EFSA J. 2009, 1181: 1 61. [2] FDA/CFSAN. Hazard analysis critical control point(haccp) -Appendices (Seafood)[J], Generic Import Product Specification, Update.1997, 25(4): 11. [3] GB/T 5009.212-2008, [S]. 作者简介 刘莹,,, : E-mail: lnciqly@126.com