WO2021223484A1 - 一种检测农药残留的方法和试剂盒 - Google Patents

一种检测农药残留的方法和试剂盒 Download PDF

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WO2021223484A1
WO2021223484A1 PCT/CN2021/076029 CN2021076029W WO2021223484A1 WO 2021223484 A1 WO2021223484 A1 WO 2021223484A1 CN 2021076029 W CN2021076029 W CN 2021076029W WO 2021223484 A1 WO2021223484 A1 WO 2021223484A1
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pesticide
preparation
insect
pesticides
detection
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PCT/CN2021/076029
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French (fr)
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刘龙飞
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北京中检葆泰生物技术有限公司
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Priority to EP21799600.8A priority Critical patent/EP4148419A4/en
Publication of WO2021223484A1 publication Critical patent/WO2021223484A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators

Definitions

  • the invention relates to a method for detecting pesticide residues, a kit and a preparation method thereof, and belongs to the fields of enzyme chemical analysis and biological detection.
  • Pesticide residue refers to the general term of pesticide precursors, derivatives, metabolites, degradation products and impurities remaining in the environment, organisms and food after pesticide use. According to its chemical structure, it can be roughly divided into organochlorine, organophosphorus, and amino. Formate and pyrethroids, etc. Excessive pesticide residues on crops and pesticide residue metabolites in agricultural and livestock products can enter the human body through the digestive tract, causing varying degrees of damage to the human body and affecting people's health.
  • Chromatography includes gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry, etc.
  • gas chromatography technology has greatly promoted the development of pesticide residue analysis and effectively improved pesticide residue analysis Detection level; as the fastest growing and most widely used analytical technology, high-performance liquid chromatography can effectively separate and detect high boiling point, poor thermal stability, and polar pesticides and their metabolites that cannot be analyzed by gas chromatography.
  • the rapid detection method has the advantages of simple operation, short detection time, low cost, and no need for large-scale instruments, and is suitable for rapid screening by corporate laboratories and grassroots regulatory agencies.
  • the current rapid detection methods mainly include chemical rapid detection method, immunoassay method, enzyme inhibition method and so on.
  • the sensitivity of rapid detection methods is low, and the sensitivity of many targets cannot meet the national limit requirements and cannot be applied to a wide range of sample types.
  • the present invention provides a rapid detection method with high sensitivity and suitable for detecting a variety of pesticides and a kit for the method.
  • One aspect of the present invention is based on the study of insect head preparations. There are certain substances in insects that can be inhibited by one or more pesticides. However, the head of an insect has many functions such as vision and touch, and has more macromolecular substances that are sensitive to pesticides. Therefore, the present invention chooses to use insect head preparations for detection experiments.
  • Another aspect of the present invention is based on accidental discoveries in actual work. Over the years, the inventor has tested the residues of various pesticides in various foods based on the principles of biological enzyme reaction and fluorescence detection. During this period, many biological materials of different species have been used, such as housefly, silkworm, beetle or bee, etc. I was surprised to find that there is a kind of bee, Apis andreniformis, whose head preparation is extremely sensitive to many different kinds of pesticides, such as organophosphorus and carbamate pesticides and their metabolites. Significantly more than other biological materials. In particular, it is extremely sensitive to profenofos, phorate, chlorpyrifos, thiocarb, dichlorvos and/or carbaryl.
  • the present invention further provides a kit and method for sensitive and rapid detection of organophosphorus and carbamate pesticides and their metabolites.
  • a high-sensitivity detection method for one or more pesticides of profenofos, phorate, chlorpyrifos, methioncarb, dichlorvos, and/or carbaryl are provided.
  • the present invention further provides an optimized and stable detection kit and detection method.
  • the invention can be widely applied to the detection of pesticide residues in foods such as cereals, dairy products, infant foods and/or meat products.
  • the kit of the present invention includes a fluorescein-containing preparation, a luminescent reagent, and an insect head preparation, which contains an enzyme that can be inhibited by one or more pesticides.
  • the insect is one or more of housefly, silkworm, beetle or bee.
  • the insect is Apis and reniformis.
  • the fluorescein-containing preparation is D-fluorescein ethyl ester.
  • the inventor found that the preparation of the black honeybee head can react with D-fluorescein ethyl ester with high specificity. Therefore, the use of D-fluorescein ethyl ester instead of other fluorescein-containing preparations as the substrate can make the detection sensitivity improve.
  • the kit of the present invention includes a black honeybee head preparation, D-fluorescein ethyl ester, luminescence reagent, buffer A, buffer B, pesticide-free residual water, and positive quality control;
  • the head preparation of the black bee contains enzymes that can be inhibited by one or more pesticides.
  • the head preparation, D-fluorescein ethyl ester and luminescent reagent of the black honeybee are all tablets.
  • the reagent is made into a compressed tablet form to ensure the stability and consistency of the reagent.
  • the kit of the present invention includes black honeybee head preparation tablets, D-fluorescein ethyl ester tablets, luminescence reagent tablets, buffer A, buffer B, and no Pesticide residual water, positive quality control tablets; the head preparation of the black bee contains enzymes that can be inhibited by one or more pesticides.
  • the present invention provides a method for detecting pesticide residues, including the use of insect head preparations containing enzymes that can be inhibited by pesticides.
  • the insect may be one or more of housefly, silkworm, beetle or honeybee; preferably, the insect is a black honey bee.
  • the pesticide is one or more of organophosphorus and/or carbamate pesticides; more preferably, the pesticide is profenofos, phorate, chlorpyrifos, metthiocarb, dichlorvos and/or carbamate One or more of Wei.
  • the present invention provides a method for detecting pesticide residues, including the use of fluorescein-containing preparations, luminescent reagents, and insect head preparations, the insect head preparations containing a pesticide that can be inhibited by one or more pesticides Enzyme.
  • the head preparation, D-fluorescein ethyl ester and luminescent reagent of the black honey bee are all tablets.
  • the method of the present invention includes the use of black honey bee head preparation tablets, D-fluorescein ethyl ester tablets, luminescent reagent tablets, buffer A, buffer B, and no pesticide residues.
  • Water, positive quality control tablets; the head preparation of the black bee contains enzymes that can be inhibited by one or more pesticides.
  • the method of the present invention includes the use of the kit of the present invention.
  • Figure 1 40% inhibition rate of different kinds of bees to pesticides.
  • the upper fold line is the result of the Italian bee
  • the middle fold line is the result of the Chinese bee
  • the lower fold line is the result of the black bee.
  • Figure 2 The relationship between D-fluorescein ethyl ester and RLU.
  • Figure 3 The relationship between incubation temperature and RLU.
  • Figure 4 The relationship between reaction time and RLU.
  • Figure 5 The relationship between incubation time and RLU.
  • a 0.07M phosphate buffer was prepared to make its pH 7.0, and EDTA and phenylthiourea were added to make their concentrations 1mM and 1 ⁇ M, respectively.
  • step b) Mix 0.5 g of the sample in step a) with 2 mL of sodium sulfate solution, and then add 2 mL of ethyl acetate-acetone solution. Take 2mL sodium sulfate and 2mL ethyl acetate-acetone solution as zero quality control. Centrifuge at 1200 ⁇ 200g for two minutes. Remove 50 ⁇ L of the upper organic phase and blow dry with nitrogen at 45 ⁇ 1°C. Re-dissolve with 1mL of pesticide-free water. Mix well with a shaker.
  • Inhibition rate (%) [(A0-A1)/A0]*100
  • A0 the RLU value of zero quality control
  • A1 the RLU value of the sample to be tested.
  • the concentration of 40% inhibition rate of different kinds of organophosphorus and carbamate pesticides on the black honey bee is significantly lower than that of the Chinese honey bee and the Italian honey bee, that is, the black honey bee is very sensitive to various pesticides.
  • the black honey bee exhibited extremely significant sensitivity to profenofos, phorate, chlorpyrifos, thiocarb, dichlorvos and/or carbaryl, at 0.4, 0.5, 0.9, 2.5, 0.4, 2.3, respectively.
  • the inhibition rate was 40%.
  • the sensitivity of black honeybees to these pesticides is about 3-5 times higher than that of Chinese honeybees and Italian honeybees.
  • D-fluorescein ethyl ester when the concentration of the added D-fluorescein ethyl ester is 0.24 ⁇ M, the RLU is the highest, indicating that 0.24 ⁇ M is the highest concentration that can react with 50 ⁇ L of the black bee head preparation. Since D-fluorescein ethyl ester is diluted by 1 mL of pesticide-free water and 50 ⁇ L is taken for each reaction, the content of D-fluorescein ethyl ester in the tablet is 0.24 nmol.
  • the RLU first increases and then decreases.
  • the temperature is 34-36°C
  • the RLU reaches the highest point, and then the RLU gradually decreases. Therefore, the activity of the black honeybee head preparation is the highest at 35 ⁇ 1°C, and this temperature is the optimal incubation temperature.
  • Example 7 The best reaction time of the black bee head preparation solution and the sample mixture
  • the phorate standard was added to the milk sample to obtain a milk sample with a concentration of 10 ⁇ g/L.
  • D-fluorescein ethyl ester and luminescence reagent solution according to the above detection procedure, and finally use a photometer to determine the RLU value.
  • Each method is performed in 6 parallels, and the results are averaged. The test results are shown in Figure 4.
  • the optimal reaction time between the black bee head preparation solution and the sample mixture is 10 minutes.
  • Example 8 The best incubation time of black bee head preparation solution and D-fluorescein ethyl ester
  • the method of the present invention is used to detect 200 samples of cereals, dairy products, meat products and infant formula foods. According to the "Technical Specification for Evaluation of Fast Food Testing Methods", the testing results are shown in Table 2-5.
  • Table 5 Formula foods for infants and young children
  • the kit and the chromatograph of the present invention were used to detect 50 samples of wheat, milk, pork and infant formula milk powder (200 samples in total), and the results were compared.
  • the method of the present invention is represented by "-"; the instrument method is represented by "ND"; when the detection result is higher than the detection limit, that is, when the result of the method of the present invention is positive, use "+” Representation; the instrument method displays the specific value of the content.
  • the comparison results are shown in Table 6 to Table 9.
  • the black bee head preparation used in the present invention can react with broad-spectrum pesticides, has strong binding ability, can accurately detect broad-spectrum pesticides and their metabolites, and has very high sensitivity.
  • the substrate adopts the black bee head preparation, which is very sensitive to D-fluorescein ethyl ester, so that the detection sensitivity is higher; the reagent is made into a compressed tablet form to ensure the stability and consistency of the reagent.

Abstract

一种检测农药残留的方法和试剂盒，在检测中采用昆虫的头部制剂，昆虫的头部制剂中含有能被农药抑制的酶。该方法的灵敏度高，假阴性率为0，假阳性率低于5％，经卡方检验，检测结果与色谱仪器测定方法无显著差异。

Description

[根据细则37.2由ISA制定的发明名称]　一种检测农药残留的方法和试剂盒 技术领域
本发明涉及一种检测农药残留的方法、试剂盒及其制备方法，属于酶化学分析、生物检测领域。
背景技术
农药残留是指农药使用后残存于环境、生物体和食品中的农药母体、衍生物、代谢物、降解物和杂质的总称，按其化学结构大致可分为有机氯类、有机磷类、氨基甲酸酯类和拟除虫菊酯类等。过量残留在农作物上的农药及农畜产品中的农药残留代谢物可经消化道进入人体，对人体造成不同程度的损害，影响人们的健康。
自20世纪50年代，各国科学家就开始研究农药残留的检测方法，常用的农残检测方法包括色谱法和快速检测方法。
色谱法包括气相色谱、气相色谱-质谱联用、高效液相色谱、液相色谱-质谱联用等，其中气相色谱技术的引进极大地推动了农药残留分析的发展，有效的提高了农药残留分析检测的水平；高效液相色谱法作为目前发展最快、应用最广泛的分析技术，对于气相色谱法不能分析的高沸点、热稳定性差和极性农药及其代谢物，可以进行有效的分离检测；质谱以及色谱-质谱联用技术的应用，使农药残留分析从一种或几种农药发展到可同时测定几十种上百种不同种类的农药，实现了对农药的高通量，高灵敏度的定性、定量检测。虽然色谱类方法结果准确可靠、灵敏度高，但由于需要大型的色谱、质谱设备，检测成本高，同时对操作人员水平要求高，限制了其在农产品企业、食品企业和基层实验室的应用。
快速检测方法由于具有操作简便，检测时间短，成本低，无需大型仪器等优势，适合于企业实验室、基层监管部门的快速筛查。目前的快速检测方法主要包括化学速测法、免疫分析法、酶抑制法等。然而，快速检测方法的灵敏度较低，许多目标物的灵敏度无法满足国家限量要求，并且不能适用于广泛的样品类型。
所以，当前存在着这样的矛盾：一方面对农药残留量进行检测属于微量或痕量分析实验,必须采用高灵敏度的检测设备才能实现；另一方面，在大规模检测的企业实验室、基层监管部门中需要采用经济便捷的方法来达到高灵敏度的效果。 通常，对于提高检测灵敏度的试验而言，研究人员一般会考虑采用进一步纯化的试剂或样品、优化试验步骤、或尝试调整检测过程中的某个或某几个参数来试图获得期望改进的效果。然而，在很多实际情况下，检测农药残留的需求不只是要检测某一种单独的农药，可能需要快速检测两种以上的农药残留情况。因此，需要探索一种灵敏度高、且适用于检测多种农药的快速检测方法。
发明内容
为了弥补现有检测方法的不足，本发明提供一种灵敏度高、且适用于检测多种农药的快速检测方法及用于该方法的试剂盒。
本发明一方面基于对昆虫头部制剂的研究。昆虫体内存在某些物质，它们能被一种或多种农药抑制。然而，昆虫的头部由于具备视觉、触觉等多种功能，具有更多的对农药敏感的大分子物质，因此，本发明选择采用昆虫的头部制剂进行检测实验。
本发明另一方面基于在实际工作中的偶然的发现。多年来，发明人基于生物酶反应和荧光检测的原理对各种食品中的多种农药残留的情况进行了检测，期间采用过很多不同物种的生物材料，例如家蝇，蚕，甲虫或蜜蜂等，惊讶地发现，有一种蜜蜂，即黑小蜜蜂(Apis andreniformis)，其头部制剂对多个不同种类的农药异常敏感，例如有机磷和氨基甲酸酯类农药及其代谢物等，且敏感性显著超过其他生物材料。尤其是，其对丙溴磷、甲拌磷、毒死蜱、甲硫威、敌敌畏和/或甲萘威格外敏感。
因此，本发明进一步提供灵敏、快速地检测有机磷和氨基甲酸酯类农药及其代谢物的试剂盒和方法。
尤其是，提供一种针对丙溴磷、甲拌磷、毒死蜱、甲硫威、敌敌畏和/或甲萘威的中一种或多种农药的高灵敏度检测方法及用于该方法的试剂盒。
本发明还进一步提供优化的、稳定的检测试剂盒和检测方法。
本发明能广泛适用于谷物、乳制品、婴幼儿食品和/或肉制品等食品中农药残留的检测。
本发明的试剂盒包括含荧光素的制剂、发光试剂和昆虫的头部制剂，所述昆虫的头部制剂含有能被一种或多种农药抑制的酶。
可选的，所述昆虫为家蝇，蚕，甲虫或蜜蜂等的一种或多种。
优选的，所述昆虫为黑小蜜蜂(Apis andreniformis)。
在一种优选的实施方式中，所述含荧光素的制剂为D-荧光素乙酯。发明人发现，黑小蜜蜂头部的制剂能高特异性地与D-荧光素乙酯反应，因此，采用D-荧光素乙酯而非其他含荧光素的制剂作为底物，能使检测灵敏度提高。
在一种实施方式中，本发明的试剂盒包括黑小蜜蜂的头部制剂、D-荧光素乙酯、发光试剂、缓冲液A、缓冲液B、无农药残水、阳性质控；所述黑小蜜蜂的头部制剂含有能被一种或多种农药抑制的酶。
在一种优选的实施方式中，所述黑小蜜蜂的头部制剂、D-荧光素乙酯和发光试剂均为片剂。将试剂制成压缩片剂的形式，能保证试剂的稳定性和一致性。
因此，在一种特别优选的实施方式中，本发明的试剂盒包括黑小蜜蜂头部制剂片剂、D-荧光素乙酯片剂、发光试剂片剂、缓冲液A、缓冲液B、无农药残水、阳性质控片剂；所述黑小蜜蜂的头部制剂含有能被一种或多种农药抑制的酶。
实际上，本发明提供了一种检测农药残留的方法，包括采用昆虫的头部制剂，所述昆虫的头部制剂中含有能被农药抑制的酶。
所述昆虫可为家蝇，蚕，甲虫或蜜蜂等的一种或多种；优选的，所述昆虫为黑小蜜蜂。
所述农药为有机磷和/或氨基甲酸酯类农药中的一种或多种；更优选的，所述农药为丙溴磷、甲拌磷、毒死蜱、甲硫威、敌敌畏和/或甲萘威中的一种或多种。
更具体的，本发明提供一种检测农药残留的方法，包括采用含荧光素的制剂、发光试剂和昆虫的头部制剂，所述昆虫的头部制剂含有能被一种或多种农药抑制的酶。
优选的，所述黑小蜜蜂的头部制剂、D-荧光素乙酯和发光试剂均为片剂。
在一种特别优选的实施方式中，本发明的方法包括采用黑小蜜蜂头部制剂片剂、D-荧光素乙酯片剂、发光试剂片剂、缓冲液A、缓冲液B、无农药残水、阳性质控片剂；所述黑小蜜蜂的头部制剂含有能被一种或多种农药抑制的酶。
可见，本发明的方法包括采用本发明的试剂盒。
附图说明
图1不同种类蜜蜂对农药的40％抑制率。上方折线为意大利蜜蜂的结果，中 间折线为中华蜜蜂的结果，下方折线为黑小蜜蜂的结果。
图2 D-荧光素乙酯与RLU的关系。
图3孵育温度与RLU的关系。
图4反应时间与RLU的关系。
图5孵育时间与RLU的关系。
具体实施方式
下面通过实施例来具体说明本发明的方法和试剂盒。本领域内的技术人员将容易理解，本申请涵盖的范围不限于说明书所描述的方法、试剂盒、和步骤等的具体实施例，据此进行同类替换和已知的改良手段均属于本发明公开的内容。
实施例1 试剂制备
一、黑小蜜蜂头部制剂制备：
1)收集黑小蜜蜂头，将其置于含大约15mL冰冷0.07M磷酸盐缓冲液(pH 7.0，含1mM EDTA和1μM苯基硫脲)的50mL烧杯中。将黑小蜜蜂头部在均质器中100rpm缓慢均质。
2)将均质物转移到离心管中3000±100rpm离心，收集上清液。
3)将上清液加入用0.07M磷酸盐缓冲液(pH 7.0，含1mM EDTA和1M苯基硫脲)平衡的sephadex G-25柱上。每2分钟收集一次馏分，以荧光素乙酸酯为底物、ATP和荧光素酶为发光剂检测酯酶的活性，并测定其生物发光。合并具有酯酶活性的部分即得。可选地，将其进一步制成含惰性填料的压缩片剂形式，作为农药残留检测中使用的黑小蜜蜂头部制剂，每片片剂中蛋白含量为4-20mg。
二、D-荧光素乙酯的制备：
称量74ng D-荧光素乙酯，可选的，将其制成含惰性填料的压缩片剂形式，每片片剂中D荧光素乙酯的含量为0.24nmol。
三、发光试剂的制备：
1)称量328.65ng ATP、13.146μg硫酸镁和10μg荧光素酶，混匀。
2)可选的，将其制成含惰性填料的压缩片剂形式，作为农药残留检测中使用的发光试剂。
四、缓冲液A制备：
制备0.07M磷酸盐缓冲液，使其pH为7.0，加入EDTA和苯基硫脲，使其浓度分别为1mM和1μM。
五、缓冲液B制备：
称量4.48g三甲基氨基酸、0.6g硫酸镁，0.146g EDTA、100mg牛血清白蛋白，77mg二硫苏糖醇，加入600mL水中。用10％的氢氧化钠将pH调整到7.8，并用蒸馏水定容至1升。
实施例2 样品制备
一、灭菌奶样品
移取100μL牛奶至1.0mL无农药水中，混匀。
二、原奶样品
移取1mL原奶至离心管，85±2℃加热5分钟，迅速冷却至0-4℃。移取100μL牛奶至1.0mL无农药水中，混匀。
三、谷物/肉制品样品
a)对于谷物：充分粉碎或研磨100g谷物样品；对于肉制品，取50g瘦肉组织，并加入食品搅拌/研磨机。加入100mL硫酸钠溶液，并高速搅拌1分钟。
b)将0.5g步骤a)中的样品与2mL硫酸钠溶液混合，然后加入2mL乙酸乙酯-丙酮溶液。以2mL硫酸钠和2mL乙酸乙酯-丙酮溶液为零质控。1200±200g离心两分钟。移取50μL上层有机相，在45±1℃用氮气吹干。用1mL无农残水重新溶解。用振荡器混合均匀。
四、婴儿食品样品
将1g婴儿食品与1mL无农残水充分混合，然后加入2mL乙酸乙酯-丙酮溶液，在1200±200g离心3分钟。以2mL无农药水和2mL乙酸乙酯-丙酮溶液为零质控。取50μL上层有机相氮气吹干。用0.5mL无农药水中重新溶解。用振荡器混合均匀。
实施例3 检测过程
一、检测步骤
1)制备黑小蜜蜂头部制剂溶液：在一个洁净的微管中，加入1mL缓冲液A 和一个黑小蜜蜂头部制剂药片，混匀。静置30秒，使固体物质沉降到微孔底部。
2)将50μL制备好的黑小蜜蜂头部制剂溶液加入100μL样品或质控中，用振荡器混匀3次。35±1℃孵育10分钟。
3)在孵育8分钟时，制备D-荧光素乙酯溶液：在一个洁净的微管中，加入1mL无农药水和一片D-荧光素-乙酯药片，混匀。静置30秒，使固体物质沉降到微孔底部。
4)10分钟孵育完成后，在孵育器中直接向每个微管底部加入50μL配好的D-荧光素乙酯溶液，混匀后，继续在35±1℃孵育5分钟。
5)孵育开始后立即制备发光试剂：将10mL缓冲液B与一片发光试剂药片混匀后静置，使固体物质沉降。
6)5分钟孵育结束时，将试管由孵育器中取出，加入1mL发光试剂，盖好微管盖。
7)用生物荧光快速检测仪读取数据，并依次记录每个微管的RLU值。
二、结果计算
按照公式计算抑制率(％)：抑制率(％)＝[(A0-A1)/A0]*100
式中：A0：零质控的RLU值；A1：待测样品的RLU值。
三、结果判定
当样品的抑制率＜40％时，表明样品中的农药为阴性。
当样品的抑制率≥40％时，表明样品中的农药为阳性。此时，可根据实际需要，采用其他方法(如色谱法)对其做进一步复检验证或确证农药的具体种类和精确含量。
实施例4 黑小蜜蜂对多种农药的超高敏感性
为了直观地体现黑小蜜蜂对农药的特殊敏感性，本实施例没有选择其他昆虫物种，而是直接选择与其生物学分类最接近的中华蜜蜂、意大利蜜蜂与黑小蜜蜂进行平行实验，按照实施例1中的“黑小蜜蜂头部制剂制备”方法制备蜜蜂头部制剂，按照上述检测步骤检测无农药水的农药加标样品，同时检测零质控，计算对蜜蜂头部制剂中的酯酶活性具有40％抑制率。检测结果见表1和图1(图1原本 为彩图，之后经过了灰度处理)。
可见，不同种类有机磷和氨基甲酸酯类农药对黑小蜜蜂的40％抑制率的浓度均显著低于中华蜜蜂和意大利蜜蜂的，即黑小蜜蜂对各类农药非常敏感。尤其是，黑小蜜蜂对丙溴磷、甲拌磷、毒死蜱、甲硫威、敌敌畏和/或甲萘威表现出极其显著地敏感性，分别为在0.4、0.5、0.9、2.5、0.4、2.3μg/kg的浓度时表现出40％的抑制率。与这些农药对中华蜜蜂和意大利蜜蜂构成40％的抑制率的相应浓度比较，黑小蜜蜂对这些农药的敏感性比中华蜜蜂和意大利蜜蜂大约高出3-5倍。
表1 40％抑制率下的农药种类及浓度
Figure PCTCN2021076029-appb-000001
实施例5 D-荧光素乙酯的最佳浓度
将100μL零质控与50μL黑小蜜蜂头部制剂溶液混匀，加入50μLD-荧光素乙酯，浓度分别为0.12μM、0.16μM、0.20μM、0.24μM、0.28μM、0.32μM和0.36μM，孵育5分钟，然后加入发光试剂溶液，立即使用光度计读取RLU值，每个浓度测定6次，取平均值。检测结果见图2。
可见，当加入的D-荧光素乙酯的浓度为0.24μM时，RLU最高，表明0.24μM是能够与50μL黑小蜜蜂头部制剂反应的最高浓度。由于D-荧光素乙酯被1mL无农药水稀释，每次反应取50μL，因此药片中D-荧光素乙酯的含量为0.24nmol。
实施例6 最佳孵育温度
将100μL零质控与50μL黑小蜜蜂头部制剂溶液混匀，加入50μLD-荧光素乙酯，分别在30℃、32℃、34℃、36℃、38℃、40℃孵育5分钟，然后加入发光试剂溶液，立即使用光度计读取RLU值，每个温度测定6次，取平均值。检测结果见图3。
可见，随温度的升高，RLU先升高后降低，当温度为34-36℃时，RLU到达最高点，随后RLU逐渐降低。因此，35±1℃时黑小蜜蜂头部制剂的活性最高，此温度为最佳孵育温度。
实施例7 黑小蜜蜂头部制剂溶液与样品混合液的最佳反应时间
向牛奶样品中加入甲拌磷标准品，获得浓度为10μg/L的牛奶样品。将100μL样品与50μL黑小蜜蜂头部制剂溶液混合，分别孵育6分钟、8分钟、10分钟、12分钟和14分钟。然后按照上述检测流程加入D-荧光素乙酯和发光试剂溶液，最后使用光度计测定RLU值，每种方法做6个平行，结果取平均值。检测结果见图4。
可见，当温度为35℃时，随孵育时间的延长，RLU逐渐降低，当孵育10分钟后，RLU下降不明显，表明孵育10分钟时，反应已经完成。因此，黑小蜜蜂头部制剂溶液与样品混合液的最佳反应时间为10分钟。
实施例8 黑小蜜蜂头部制剂溶液与D-荧光素乙酯的最佳孵育时间
将100μL零质控与50μL黑小蜜蜂头部制剂溶液混匀，加入50μL D-荧光素 乙酯，分别在35℃孵育2分钟、3分钟、4分钟、5分钟、6分钟、7分钟和8分钟，然后加入发光试剂溶液，立即使用光度计读取RLU值，每个温度测定6次，取平均值。检测结果见图5。可见，最佳孵育时间为5分钟。
实施例9 本发明方法的假阴性率、假阳性率和特异性
采用本发明的方法分别对200份谷物、乳制品、肉制品和婴幼儿配方食品样品进行检测，根据《食品快速检测方法评价技术规范》，检测结果见表2-5。
表2 谷物
Figure PCTCN2021076029-appb-000002
表3 乳制品
Figure PCTCN2021076029-appb-000003
表4 肉制品
Figure PCTCN2021076029-appb-000004
表5 婴幼儿配方食品
Figure PCTCN2021076029-appb-000005
由表2-5可见，对于不同样品，假阴性率均为0，假阳性率均小于5％，特异性均大于95％。
实验例10 本发明的试剂盒与色谱类仪器测定结果的一致性
分别用本发明的试剂盒与色谱仪检测小麦、牛奶、猪肉和婴幼儿配方奶粉各50份样品(共200份)，并将结果进行对比。
当检测结果低于检测限时，本发明所述方法用“-”表示；仪器方法用“ND”表示；当检测结果高于检测限，即本发明所述方法结果为阳性时，用“+”表示；仪器方法显示含量具体数值。其对比结果如表6-表9所示。
表6 本发明所述方法与仪器方法对比结果(小麦)(μg/kg)
Figure PCTCN2021076029-appb-000006
表7 本发明所述方法与仪器方法对比结果(牛奶)(μg/kg)
Figure PCTCN2021076029-appb-000007
表8 本发明所述方法与仪器方法对比结果(猪肉)(μg/kg)
Figure PCTCN2021076029-appb-000008
表9 本发明所述方法与仪器方法对比结果(婴幼儿配方食品)(μg/kg)
Figure PCTCN2021076029-appb-000009
由表6-9可见，色谱仪器检测出19份阳性样品，该19份样品经本发明的试剂盒检测，结果也均为阳性；色谱仪检测出阴性样品181份，该181份样品经本发明的试剂盒检测后显示，178份为阴性，3份为阳性。即本方法灵敏度高而无假阴性。
表10 本发明所述方法与仪器方法实验数据分布表
Figure PCTCN2021076029-appb-000010
对上述两种方法的检测结果进行卡方检验，由表10可计算出显著性差异(χ 2)为：
χ 2＝(|0-3|-1) 2/(0+3)＝1.33
χ 2＜3.84，表示本发明的试剂盒与色谱仪检测的阳性确证比率在95％的置信区间内无显著性差异。
综上，本发明所采用的黑小蜜蜂头部制剂能与广谱的农药反应，结合能力强，能准确检测广谱的农药及其代谢物，且灵敏度非常高。优选的，底物采用黑小蜜蜂头部制剂非常敏感的对D-荧光素乙酯，使检测灵敏度更高；将试剂制成压缩片剂的形式，能保证试剂的稳定性和一致性。
虽然已经详细说明了本发明及其优点，但是应当理解在不超出由所附的权利要求所限定的本发明的精神和范围的情况下可以进行各种改变、替代和变换。

Claims (10)

  1. 一种试剂盒，包括含荧光素的制剂、发光试剂和昆虫的头部制剂，其特征在于，所述昆虫的头部制剂中含有能被农药抑制的物质。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述昆虫为蜜蜂；优选的，所述蜜蜂为黑小蜜蜂。
  3. 根据权利要求2所述的试剂盒，其特征在于，所述的农药为有机磷和/或氨基甲酸酯类农药中的一种或多种。
  4. 根据权利要求3所述的试剂盒，其特征在于，所述农药为丙溴磷、甲拌磷、毒死蜱、甲硫威、敌敌畏和/或甲萘威中的一种或多种。
  5. 根据权利要求1-4之一所述的试剂盒，其特征在于，所述含荧光素的制剂为D-荧光素乙酯。
  6. 根据权利要求6所述的试剂盒，其特征在于，所述含荧光素的制剂、发光试剂和昆虫的头部制剂均为片剂。
  7. 权利要求1-6之一所述的试剂盒在检测农药残留中的用途。
  8. 一种检测农药残留的方法，包括在检测中采用昆虫的头部制剂，所述昆虫的头部制剂中含有能被农药抑制的酶。
  9. 根据权利要求8所述的方法，其特征在于，所述昆虫为蜜蜂；优选的，所述昆虫为黑小蜜蜂。
  10. 根据权利要求8或9所述的方法，其特征在于，所述农药为有机磷和/或氨基甲酸酯类农药；更优选的，所述农药为丙溴磷、甲拌磷、毒死蜱、甲硫威、敌敌畏和/或甲萘威中的一种或多种。
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