胶体金免疫层析法快速检测蔬菜中3 种农药残留

doi:10.15933/j.cnki.1004-3268.2024.06.009

摘要/Abstract

摘要： 基于胶体金免疫层析原理研制吡虫啉、丙溴磷及克百威快速检测试纸条，评价其在大蒜、白菜检测中的应用效果。采用柠檬酸三钠还原法制备胶体金，用胶体金颗粒标记吡虫啉、丙溴磷及克百威单克隆抗体作为检测探针，以硝酸纤维素膜为固相载体，包被吡虫啉-BSA、丙溴磷-BSA及克百威-BSA偶联物为检测线，羊抗鼠IgG抗体为质控线，制备胶体金免疫层析试纸条，并对金标抗体用量、标记抗体浓度、检测线包被浓度等参数条件进行优化，评价试纸条的灵敏度、特异性、稳定性和准确性。以大蒜、白菜为对象，优化了样品前处理方法，并对这2种蔬菜样品进行阴性检测及添加标准品农药检测。结果表明，优化后的工艺参数中吡虫啉、丙溴磷、克百威的最适标记抗体质量浓度分别为9.6、8.4、9.6 μg/mL，抗原的包被质量浓度均是0.3 mg/mL，检测用时约15 min。吡虫啉、丙溴磷及克百威的检出限分别为20、3 000、20 ng/mL，符合国标对2种蔬菜的限量规定。检测与吡虫啉、丙溴磷及克百威功能和结构相似的其他药物时无交叉反应，特异性较好。克百威和吡虫啉农药的最适提取剂分别为4 mL乙酸乙酯和4 mL甲醇，样品用量均为2 g，上清液3 mL；丙溴磷农药的最适提取剂是9 mL乙酸乙酯，样品用量3 g，上清液8 mL，上清液用氮气吹干。50份蔬菜阴性样品和加标样品的检测结果显示，假阳性率为6%，假阴性率为0。可见，研制的试纸条适用于大蒜、白菜现场快速检测以及基层实验室中大量样本的筛查，可为大蒜、白菜中农药残留控制提供有效监管手段。

关键词: 大蒜, 白菜, 胶体金快速检测, 吡虫啉, 丙溴磷, 克百威

Abstract: Rapid test strips for the detection of imidacloprid,profenofos and carbofuran were developed based on colloidal gold immunochromatography,and its application effect in the detection of garlic and cabbage was evaluated.The test strips for rapid detection of imidacloprid,profenofos and carbofuran were established by preparation of colloidal gold by reduction with trisodium citrate,with the monoclonal antibodies of imidacloprid,profenofos and carbofuran labeled with colloidal gold particles as the detection probes,nitrocellulose membrane as the solid‑phase carrier,imidacloprid‑BSA,profenofos‑BSA and carbofuran‑BSA conjugate as the detection lines,and goat anti‑mouse IgG antibody as the control line.The parameter conditions,including gold‑labeled antibody usage,concentrations of antibody for labeling,and concentration of the coating antigen were optimized. Under the optimal condition,the sensitivity,specificity and stability,as well as the accuracy of the test strips were evaluated.With optimization of sample extraction,the test strips were employed for the detection of pesticides in two vegetable samples by negative detection and addition of standardized pesticide products.The results showed that in the optimized process parameters，the concentrations of colloidal gold labeled antibody of imidacloprid,profenofos and carbofuran were 9.6,8.4,9.6 μg/mL,respectively,the concentrations of the antigens coated were all 0.3 mg/mL,and the detection time was about 15 min.The limits of detection of imidacloprid,profenofos and carbofuran were 20,3 000,and 20 ng/mL,which were in compliance with the limits of the national standard for the two kinds of vegetables.There was no cross‑reactivity and good specificity in the detection of other drugs with similar function and structure as imidacloprid,profenofos and carbofuran.The most suitable extractants for carbofuran and imidacloprid pesticides were 4 mL of ethyl acetate and 4 mL of methanol,respectively,with a sample size of 2 g and a supernatant of 3 mL;for profenofos pesticide,the most suitable extractant was 9 mL of ethyl acetate,with a sample size of 3 g and a supernatant of 8 mL,and the supernatant was dried with nitrogen gas.The detection results of 50 negative and spiked samples of vegetables showed a false positive rate of 6% and a false negative rate of 0.It can be seen that the developed test strips are suitable for on‑site rapid detection of garlic and cabbage as well as screening of a large number of samples in grass‑roots laboratories,and can provide an effective regulatory tool for the control of pesticide residues in garlic and cabbage.

Key words: Garlic, Cabbage, Colloidal gold rapid detection, Imidacloprid, Profenofos, Carbofuran

中图分类号:

S481⁺.8

魏茂琼, 王丽, 兰珊珊, 刘振环, 沙凌杰, 林昕, 李俊, 普娅丽, 刘宏程, . 胶体金免疫层析法快速检测蔬菜中3 种农药残留[J]. 河南农业科学, 2024, 53(6): 79-90.

WEI Maoqiong, WANG Li, LAN Shanshan, LIU Zhenhuan, SHA Lingjie, LIN Xin, LI Jun, PU Yali, LIU Hongcheng. Rapid Detection of Three Pesticide Residues in Vegetables Using Colloidal Gold Immunoassay[J]. Journal of Henan Agricultural Sciences, 2024, 53(6): 79-90.

参考文献

［1］张秀花，苑婷婷，李季，等.蔬菜中农药残留快速检测与定量检测结果符合性分析［J］.食品安全质量检测学报，2020，11（4）：1281‑1287.
ZHANG X H，YUAN T T，LI J，et al.Coincidence analysis of rapid detection and quantitative detection results of pesticide residues in vegetables［J］.Journal of Food Safety & Quality，2020，11（4）：1281‑1287.
［2］韩曦莹.气相色谱法测定陈皮中丙溴磷的残留量［J］.食品安全质量检测学报，2020，11（10）：3301‑3305.
HAN X Y.Determination of profenofos in tangerine peel by gas chromatograph［J］.Journal of Food Safety &Quality，2020，11（10）：3301‑3305.
［3］LI Z X，ZHANG Y H，ZHAO Q Y，et al.Occurrence，temporal variation，quality and safety assessment of pesticide residues on citrus fruits in China［J］.Chemosphere，2020，258：127381.
［4］刘刚.应高度重视丙溴磷在我国柑橘中的残留问题［J］.农药市场信息，2019（12）：37‑39．
LIU G.Attention should be paid to the residue of probromophos in Chinese citrus［J］.Pesticide Market News，2019（12）：37‑39.
［5］国家卫生健康委员会，农业农村部，国家市场监督管理总局.食品安全国家标准食品中农药最大残留限量：GB 2763—2021［S］.北京：中国标准出版社，2021.
National Health Commission，Ministry of Agriculture and Rural Development，State Administration for Market
Supervision and Administration.National Standard for Food Safety Maximum Residue Limits of Pesticides in Food：GB 2763—2021［S］.Beijing：China Standard Press，2021.
［6］European Commission. Pesticides EU‑MRLs［DB/OL］.（2020‑11‑13）［2022‑04‑28］.http：//ec.europa.eu/food/plant/pesticides/eu‑pesticides‑database.
［7］The Japan Food Chemical Research Foundation.Positive list system for agricultural chemical residues in foods［DB/OL］.（2015‑04‑08）［2022‑04‑10］.http：//db. ffcr.or.jp/front
［8］中国农业工业协会. 2016年国家禁用和限用的农药名录汇总［J］.今日农药，2017（2）：9.
China Agricultural Industry Association.List of pesticides banned and restricted by the state in 2016［J］.PesticidesToday，2017（2）：9.
［9］李俊辉，韩煜晖，桂赟，等.普通白菜中4种常用农药残留及风险评估［J］.现代农业科技，2018（4）：117‑118.
LI J H，HAN Y H，GUI Y，et al.Residue and risk assessment of four pesticides in common Chinese cabbage［J］.Modern Agricultural Science and Technology，2018（4）：117‑118.
［10］梁启富，史梦竹，方灵，等.福建省主产区普通白菜中农药残留水平及膳食暴露风险评估［J］.农药学学报，2023，25（2）：453‑460.
LIANG Q F，SHI M Z，FANG L，et al.Pesticide residue levels and dietary exposure risk assessment in pak‑choi（Brassica rapa ssp.chinensis）in Fujian production area［J］.Chinese Journal of Pesticide Science，2023，25（2）：453‑460.
［11］林静，张顺，蔡挺，等.QuEChERS-超高效液相色谱‑串联质谱技术同时测定大蒜中10种农药残留［J］.浙江农业学报，2018，30（1）：159‑166.
LIN J，ZHANG S，CAI T，et al.Determination of 10 pesticide residues in garlic by QuEChERS‑ultra performance liquid chromatography‑tandem mass spectrometry［J］.Acta Agriculturae Zhejiangensis，2018，30（1）：159‑166.
［12］LI G R，YU W Q，XIAO Z J，et al.Research status of toxicity and residue detection of high‑risk pesticide adjuvants［J］.Plant Diseases and Pests，2019，10（4）：26‑30.
［13］方海仙，耿慧春，普娅丽，等.微波处理-气相色谱法测定洋葱中5种氯代酰胺类除草剂残留［J］.山西农业科学，2022，50（1）：54‑60.
FANG H X，GENG H C，PU Y L，et al.Determination of five chloroacetamide herbicides in onion using a microwave‑assisted treatment and gas chromatography method［J］.Journal of Shanxi Agricultural Sciences，2022，50（1）：54‑60.
［14］尤艳莉，李福翔，孙甲霖，等.液相色谱串联质谱法检测海产养殖用水中乙酰甲喹及其代谢物残留［J］.南方农业学报，2023，54（1）：238‑249.
YOU Y L，LI F X，SUN J L，et al.Determination of mequindox and its metabolites in mariculture water by liquid chromatography‑tandem mass spectrometry［J］.Journal of Southern Agriculture，2023，54（1）：238‑249.
［15］HUANG，HUABIN，ZHOU，et al.A device for rapid detection of dithiocarbamate pesticide residues in fruits［J］. Plant Diseases and Pests，2014，5：39‑41.
［16］李萍萍，吴小芳，万瑶，等.固相萃取-气相色谱-串联质谱法测定大蒜中19种有机磷农药残留量［J］.农药学学报，2014，16（6）：720‑727.
LI P P，WU X F，WAN Y，et al.Determination of 19 organophosphorus pesticide residues in garlic using solid‑phase extraction and gas chromatography‑tandem mass spectrometry［J］.Chinese Journal of Pesticide Science，2014，16（6）：720‑727.
［17］尹超，李晋.水果和蔬菜中农药残留检测技术研究进展［J］.现代食品，2022，28（1）：38‑40.
YIN C，LI J.Research progress in detection of pesticide residues in fruits and vegetables［J］.Modern Food，2022，28（1）：38‑40.
［18］贺燕，王益军，张苏珍，等.禽蛋中呋喃唑酮代谢物（AOZ）残留量的酶联免疫检测［J］.中国动物保健，2021，23（8）：113‑115.
HE Y，WANG Y J，ZHANG S Z，et al.Enzyme‑linked immunosorbent assay for furazolidone metabolite（AOZ）residues in poultry eggs［J］.China Animal Health，2021，23（8）：113‑115.
［19］龚航，刘贝贝，李盼，等.基于三唑磷残留限量值的多检测线免疫试纸条的制备与应用［J］.分析化学，2018，46（6）：938‑946.
GONG H， LIU B B， LI P， et al.Immunochromatographic assay for maximal residual limit‑based multiple quantitative determination of triazophos in agro products［J］.Chinese Journal of Analytical Chemistry，2018，46（6）：938‑946.
［20］姚蕾珺.吡虫啉、啶虫脒、克百威及涕灭威的免疫快速检测［D］.无锡：江南大学，2017.
YAO L J.Development of rapid immunoassay for imidacloprid，acetamiprid，carbofuran and aldicarb［D］.Wuxi：Jiangnan University，2017.
［21］曹涤非，薛佳莹，崔向红，等.柠檬酸三钠法制备胶体金及其应用［J］.化学工程师，2019，33（12）：12‑14.
CAO D F，XUE J Y，CUI X H，et al.Preparation and application of colloidal gold by trisodium citrate［J］.Chemical Engineer，2019，33（12）：12‑14.
［22］金洪伟，裴小龙，陈发荣，等.胶体金免疫层析法检测果蔬中毒死蜱前处理的优化［J］.粮食与油脂，2023，36（9）：141‑144.
JIN H W，PEI X L，CHEN F R，et al.Optimization of pretreatment during the detection of chlorpyrifos in fruits and vegetables by colloidal gold immunochromatography［J］.Cereals & Oils，2023，36（9）：141‑144.
［23］王忠兴.食品中九种兽药残留免疫快速检测方法研究［D］.无锡：江南大学，2019.
WANG Z X.Rapid immunoassay for testing nine veterinary drugs residues in food samples［D］.Wuxi：Jiangnan University，2019.
［24］YAN X J，WANG Z B，BARLOW P.Quantitative estimation of garlic oil content in garlic oil based health products［J］.Food Chemistry，1992，45（2）：135‑139.
［25］贾惠言，吴银良.柑橘中农药多残留检测方法比对分析［J］.浙江农业科学，2022，63（6）：1345‑1350.
JIA H Y，WU Y L.Comparative analysis of detection methods for multiple pesticide residues in citrus［J］.Journal of Zhejiang Agricultural Sciences，2022，63（6）：1345‑1350.
［26］WANG L M，CAI J，WANG Y L，et al.A bare‑eye‑based lateral flow immunoassay based on the use of gold nanoparticles for simultaneous detection of three pesticides［J］.Microchimica Acta，2014，181（13）：1565‑1572.
［27］叶茂，沈晓玲，陈青舟，等.胶体金免疫层析法同时检测果蔬中四种农药残留［J］.食品工业科技，2023，44（6）：300‑308.
YE M，SHEN X L，CHEN Q Z，et al.Simultaneous determination of four pesticide residues in fruits and vegetables by colloidal gold immunochromatography［J］.Science and Technology of Food Industry，2023，44（6）：300‑308.
［28］王兆芹，张富生，邓家军，等.基于单克隆抗体的胶体金免疫层析法快速检测丙溴磷［J］.河南农业科学，2020，49（12）：77‑82.
WANG Z Q，ZHANG F S，DENG J J，et al.Rapid detection of profenofos using colloidal gold immunoassay based on monoclonal antibody［J］.Journal of Henan Agricultural Sciences，2020，49（12）：77‑82.
［29］朱亮亮，王琳琛，王兆芹，等.检测4种有机磷农药胶体金免疫层析试纸条的研制［J］.山东畜牧兽医，2019，40（8）：10‑12.
ZHU L L，WANG L C，WANG Z Q，et al.Development of colloidal gold immunochromatographic test strip for detecting four organophosphorus pesticides［J］.Shandong Journal of Animal Science and Veterinary Medicine，2019，40（8）：10‑12.

[1]	张丹. 化肥减量配施生物有机肥和灌溉量对大蒜产量和品质的影响[J]. 河南农业科学, 2022, 51(3): 139-145.
[2]	马嘉俊, 吴英华, 李璿, 李梅兰, 侯雷平. 白菜NPF 基因家族成员的鉴定及其生物信息学分析[J]. 河南农业科学, 2021, 50(9): 117-127.
[3]	张睿, 何超, 李河, 沈登荣, 袁盛勇, 李珣. 吡虫啉对异迟眼蕈蚊抗性筛选及其对解毒酶活性的影响[J]. 河南农业科学, 2021, 50(7): 110-116.
[4]	张运红, 骆晓声, 寇长林, 郭战玲. 施用不同阻隔材料对小白菜产量、品质及氮磷吸收的影响[J]. 河南农业科学, 2021, 50(4): 115-123.
[5]	常虹, 秦广利, 王红军, 陈妍. 土壤调理剂与微生物有机肥配施对黄潮土区重茬大蒜土壤改良及产量的影响[J]. 河南农业科学, 2021, 50(11): 72-78.
[6]	苗秀荣, 来雪慧, 李梦茜, 李玉, 李娜, 冯如意. 不同钝化剂对土壤有效态重金属含量及其在小白菜中累积的影响[J]. 河南农业科学, 2020, 49(8): 63-71.
[7]	何方伟, 崔宏浩, 王文华, 朱青, 陈正刚, 张钦, 张爱华, 姚单君, 况胜剑, 杨俐苹, 杨秀海. 酒糟沼液与化肥配施对白菜产量、品质和氮肥利用效率的影响[J]. 河南农业科学, 2020, 49(7): 93-100.
[8]	闫敏, 王晗, 刘少华, 顾小敏, 许晔. 大白菜BrROP基因家族的生物信息学分析[J]. 河南农业科学, 2020, 49(4): 93-100.
[9]	王兆芹, 张富生, 邓家军, 马玉华, 崔廷婷, 崔娜, 万宇平. 基于单克隆抗体的胶体金免疫层析法快速检测丙溴磷[J]. 河南农业科学, 2020, 49(12): 77-82.
[10]	苏旺苍, 郝红丹, 徐洪乐, 孙兰兰, 周蒲, 吴仁海, 薛飞. 31.9%吡虫啉·戊唑醇悬浮种衣剂对河南省主播小麦生长的影响[J]. 河南农业科学, 2019, 48(12): 89-97.
[11]	张红瑞，张云霞，李纪红，沈玉聪，高致明，张子龙. 2种单体皂苷及三七总皂苷对白菜幼苗生长生理的影响[J]. 河南农业科学, 2019, 48(1): 94-98,104.
[12]	魏小春，原玉香，赵艳艳，王志勇，杨双娟，郑小兰，段俊枝，张晓伟. 大白菜BrCOX11基因的克隆及表达分析[J]. 河南农业科学, 2018, 47(9): 93-98.
[13]	梁肖娜,叶清,吴尚,吴尚仪,康世墨,关博元,陶冬冰,岳喜庆. 一种新型鸡血液体肥料的肥效试验研究[J]. 河南农业科学, 2018, 47(2): 58-62.
[14]	吴涛，董彦琪，肖艳，王晓玲，原连庄，原让花，孙玉镯，周俊国. 大白菜主要表型性状的配合力评价及遗传力分析[J]. 河南农业科学, 2018, 47(12): 102-109.
[15]	刘爱芝，韩松，郭小奇，梁九进，杨春英,曹秀，鲁传涛. 3代新烟碱类杀虫剂对小麦蚜虫的防治效果比较[J]. 河南农业科学, 2017, 46(9): 83-87.