Application of Time-resolved Fluorescence Immunoassay to Concurrently Quantify Fumonisin B1, B2 and B3 in Corn
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摘要: 建立了一种可同时定量检测玉米中伏马毒素B1、B2和B3的时间分辨荧光免疫层析方法。利用活化酯法制备时间分辨荧光微球和伏马毒素抗体的偶联物,将伏马毒素抗原包被到硝酸纤维素膜上作为T线,通过优化不同T线包被条数,最终建立伏马毒素竞争性免疫层析方法。并对所建立方法的灵敏度、特异性、准确度、精密度和与国标方法分析结果的相关性进行评价。包被两条T线建立的检测方法检出限为107.68~168.28 μg/kg,定量限为283.46~444.63 μg/kg;与伏马毒素B1、伏马毒素B2和伏马毒素B3的交叉反应率分别为100%、85.59%和72.72%,与其它5种常见的真菌毒素均无明显交叉;加标回收率范围88.37%~117.42%,变异系数小于10%;该方法与国标方法GB 5009.240-2016 中规定的免疫亲和柱净化-高效液相色谱法(IAC-HPLC)检测结果的符合度在92.17%~107.21%之间。建立的时间分辨荧光免疫层析检测方法能满足对玉米中伏马毒素B1、B2和B3的现场快速定量检测。Abstract: This study aimed to establish a time-resolved fluorescence immunoassay method for the simultaneous quantitative detection of fumonisin B1, B2 and B3 in corn. The EDC/NHS active ester method was used to prepare time-resolved fluorescent microspheres and fumonisin antibody conjugates. Fumonisin antigen was immobilized on a nitrocellulose membrane as the test line. By optimizing the number of test line, a competitive immunoassay method for detecting fumonisin was eventually established. Subsequently, the sensitivity, specificity, accuracy, precision, and correlation with the national standard method were evaluated for the established method. The detection limits of the established method by using two test line pattern were 107.68~168.28 μg/kg, and the quantification limits were 283.46~444.63 μg/kg. The cross-reactivity with fumonisin B1, fumonisin B2 and fumonisin B3 were 100%, 85.59% and 72.72%, respectively, and no significant cross-reactivity was observed with five other common mycotoxins. The recovery rates ranged from 88.37% to 117.42%, and the coefficients of variation were lower than 10%. The conformity of the results obtained by the established method, as compared to the immunoaffinity column purification-high performance liquid chromatography (IAC-HPLC) method specified in the national standard GB 5009.240-2016, fell within the range of 92.17% to 107.21%. The established time-resolved fluorescence immunoassay method meets the requirements for rapid on-site quantitative detection of fumonisin B1, B2 and B3 in corn.
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Keywords:
- time-resolved fluorescent microsphere /
- fumonisin B1 /
- fumonisin B2 /
- fumonisin B3 /
- corn /
- quantitative detection
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伏马毒素(Fumonisin,FB)主要由串珠镰刀菌、黑曲霉、链格孢霉菌产生的一种代谢产物,又名烟曲霉毒素。该类毒素是由不同的多氢醇和丙三羧酸组成的双酯类化合物,易溶于水,热稳定性强。已发现的天然伏马毒素共有FA、FB、FC和FP四种类型,28种不同的异构体,其中的FB族的污染占比最高,毒性也最强。FB族主要包括FB1、FB2、FB3,FB1被国际癌症研究机构(IARC)评定为2B类致癌物[1−2]。国内饲料霉菌污染调查显示2015年至2020年在玉米及加工副产品中FB1的阳性检出率均在60%以上,2020年检出率高达100%,阳性平均值大于1000 μg/kg;在2015年阳性平均值高达4500 μg/kg[3]。一份针对四川地区真菌毒素污染的调查报告也显示抽检的玉米样品中FB1、FB2、FB3均有检出,总检测率为30.56%[4]。研究表明,FB可以通过对鞘脂代谢通路和氧化应激的影响从而对实验动物产生神经毒性、免疫毒性、组织器官毒性等毒性作用[5−7]。FB也与人类的食管癌有密切关系[8]。因其危害较大,国际食品法典委员会和欧盟规定玉米中FB1和FB2总量不得超过4000 μg/kg,美国食品及药物管理局规定脱胚的干磨玉米制品中FB1、FB2和FB3不得超过2000 μg/kg[9]。我国《饲料卫生标准》中饲料玉米及其加工品中FB1与FB2含量总和不高于60 mg/kg[10]。
目前检测谷物饲料中FB的常用方法主要分为仪器分析方法和免疫学分析方法两大类[11]。其中仪器分析方法主要有高效液相色谱法(High performance liquid chromatography,HPLC),液相色谱-质谱联用(Liquid chromatography-mass spectrometry,LC-MS)法[12−14]。HPLC和LC-MS具有良好的灵敏度、选择性和重复性,但设备昂贵需特殊的清理步骤,并且检测周期长成本高不适合现场快速检测。免疫学分析方法主要有联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)[15]和胶体金侧向免疫法[16−17]。ELISA具有样本制备简单、成本低、无放射性等优点,但也存在操作步骤繁琐的缺点。胶体金侧向免疫层析可以用较低的成本,较少的实验步骤对FB进行检测,但具有灵敏度低,定量范围窄,结果随环境变化波动较大的缺点。
时间分辨荧光免疫层析(Time-resolved fluoroimmunoassay,TRFIA)方法利用铕、钐、镨等稀土元素及螯合物组成的荧光微球作为标记物标记抗原或者抗体。因其荧光衰变期长,Stokes位移大,发射峰狭窄的特点,使其可以消除各种非特异性荧光的干扰,能够实现对谷物饲料中FB的准确定量检测[18]。现有应用TRFIA方法对谷物饲料中FB检测的对象只有FB1,对FB2和FB3的检测相对较少,并且采用的均为一条T线模式,未对不同T线条数做对比测试[19−21]。本研究拟对不同T线条数做对比,选定最佳T线条数后建立能够同时检测FB1、FB2、FB3的定量检测方法,以期为更加准确全面地检测玉米中FB提供可行的检测方案。
1. 材料与方法
1.1 材料与仪器
铕元素羧基时间分辨荧光微球(365/610 nm,λex/λem) 美国Bangs Laboratories;FB单克隆抗体、FB抗原 北京维德维康生物技术有限公司;酶标二抗、鸡IgY、兔抗鸡多克隆抗体 苏州博特龙免疫技术有限公司;吐温20、甲醇 分析纯,国药集团化学试剂有限公司;硝酸纤维素膜(NC膜) 德国Sartorius;FB阳性、FB阴性玉米样本 国家粮食和物资储备局科学研究院;FB1、FB2、FB3标准品、碳二亚胺(EDC)、N-羟基硫代琥珀酰亚胺(NHS) 美国sigma-Aldrich;PVC背板、吸水纸、样品垫 上海金标生物科技有限公司;微孔板 江苏迪默生物科技有限公司。
ES520电子天平 天津德安特传感技术有限公司;XYZ3060划膜仪 美国BIODOT;Fresco21冷冻离心机 美国Thermo Fisher Scientific;KQ-100E超声波清洗仪 昆山市超声仪器有限公司;MX-F涡动仪 大龙兴创实验仪器(北京)股份公司;WH-400检测卡恒温孵育器、FQ-S3时间分辨荧光免疫定量分析仪 北京维德维康生物技术有限公司。
1.2 实验方法
1.2.1 溶液配制
荧光微球复溶液配制:Tris 12.11 g,加入适量纯水溶解后用浓盐酸调节pH为8.0,BSA 20 g,吐温20 20 g,蔗糖100 g,NaCl 9 g,Proclin 300防腐剂0.2 g,用纯水定容至1000 mL。冻干保护液配制:硼酸3.7 g,十水合四硼酸钠13.3 g,蔗糖20 g,BSA 10 g,PVP 5 g,Proclin 300防腐剂 0.2 g,用纯水定容至1000 mL。样品稀释液配制:Tris 40.38 g,加入适量纯水溶解后,用浓盐酸调节pH至8.0,加入50 g PVP,加入5 g S9,用纯水定容至1000 mL。
1.2.2 硝酸纤维素膜包被及不同包被模式测试
制备一条T线和两条T线两种包被模式的FB膜[22−23]。其中一条T线膜是将FB抗原(3.5 mg/mL)用0.02 mol/L PB缓冲液(pH7.4)2倍稀释作为包被液,用划膜仪以0.8 μL/cm的喷量包被到NC膜上。两条T线膜是将FB抗原(3.5 mg/mL)用0.02 mol/L PB缓冲液(pH7.4)4倍稀释平行包被两条T线到NC膜上。两种膜均以兔抗鸡多克隆抗体(5 mg/mL)用0.02 mol/L PB缓冲液(pH7.4)6倍稀释作为C线。将制备好的NC膜置于45 ℃ 烘箱中烘干16 h。用两种膜分别检测浓度为0、0.5、1、2、4、8 μg/kg的FB1标准品,用FQ-S3时间分辨荧光免疫定量分析仪读取各浓度点T/C及(T1+T2)/C值,每个浓度点测试三次取均值,通过公式抑制率(%)=(阴性TC比值−加标准品后TC比值)/阴性TC比值×100计算各点抑制率。根据抑制率的结果,确定最终包被模式。
1.2.3 FB及鸡IgY荧光探针制备
FB荧光探针制备[24−25]。取100 μL时间分辨荧光微球(0.19 μm,1%),加入900 μL活化缓冲液(0.05 mol/L MES,pH6.0)涡旋仪振动5 s,离心机15000×g,离心15 min,弃上清加入520 μL活化缓冲液,使用超声仪超声5 min,依次加入Sulfo-NHS(0.001 mol/L)315 μL和EDC(0.001 mol/L)165 μL,水平摇床上室温200 r/min活化15 min;之后加千分之一吐温,用离心机15000×g,离心15 min,弃上清。向沉淀中加入500 μL偶联缓冲液(0.04 mol/L BBS,pH8.0),超声仪超声5 min,使沉淀复溶。取10 μL FB单克隆抗体(6.1 mg/mL)加入到490 μL偶联缓冲液中,混匀,将混匀后液体加入到复溶后的荧光微球中,水平摇床室温200 r/min反应2 h。加千分之一吐温,离心机15000×g,离心15 min,弃上清。向沉淀中加入400 μL封闭缓冲液(0.01 mol/L PBS+2% BSA)封闭荧光微球,超声2 min,水平摇床室温200 r/min反应2 h;加万分之一吐温,用离心机15000×g,离心15 min,弃上清。使用1000 μL 荧光微球复溶液复溶微球,超声仪超声5 min,4 ℃保存。鸡IgY荧光探针制备与FB荧光探针制备步骤相同,只有在标记抗体步骤将用鸡IgY(10 mg/mL)替代FB单克隆抗体,抗体加入量100 μL。将上述FB荧光探针,鸡IgY荧光探针与冻干保护液按照1:0.1:48.9(V:V:V)充分混匀,按照50 μL/孔添加到96孔板中进行真空冷冻干燥。冻干程序为:冷冻阶段:标准大气压,−40 ℃,保持120 min。升华阶段:5 Pa,−10 ℃,再干燥阶段:5 Pa,30 ℃ 600 min[26]。
1.2.4 检测卡的组装
将制备完成的样品垫裁为长300 mm宽28 mm的规格,吸水纸裁为长300 mm宽30 mm的规格,依次粘贴到长300 mm宽80 mm的PVC背板上,每种组分衔接约2 mm,最后用裁刀裁为宽4 mm的试剂条(图1),安装到规格为长100 mm宽20 mm的荧光卡壳中(图2)。
1.2.5 样品前处理
称取粉碎后(过20目筛)的国家粮食和物资储备局科学研究院玉米样本5±0.05 g于50 mL离心管中,加入70%甲醇-水溶液25 mL,2500 r/min涡动5 min,室温4000×g离心5 min。取5 μL上清液于800 μL样品稀释液中稀释混匀,待检。
1.2.6 检测步骤
检测卡恒温孵育器调整至40 ℃,并将FB检测卡提前置于检测卡恒温孵育器中,打开FQ-S3荧光免疫定量分析仪预热。取100 μL待检样品加入到FB冻干微孔中,吹打混匀后将液体转移至FB检测卡的加样孔中,准确反应6 min,将反应结束的FB检测卡用FQ-S3时间分辨荧光免疫定量分析仪进行检测。
1.2.7 检测方法的评价
1.2.7.1 定量曲线
取国家粮食和物资储备局科学研究院玉米阴性样本,分别加入不同量的FB1、FB2、FB3标准溶液至样品含量为0、250、500、1000、2000、4000、8000 μg/kg。涡动混匀后按1.2.5进行样本前处理,再按照1.2.6所述进行检测,每个浓度水平6组平行实验。以浓度值作为横坐标,以(T1+T2)/C的均值为纵坐标,用Origin 2018软件中logistic函数进行非线性拟合。
1.2.7.2 特异性测试
以FB1为基准分别测试与FB2、FB3、黄曲霉毒素B1、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、赭曲霉毒素A、T-2毒素的交叉反应率(Cross-reactivity,CR)。具体方法为按1.2.7.1所述方式分别绘制上述霉菌毒素的定量曲线,根据标准曲线分别拟合得出半抑制浓度(Half maximal inhibitory concentration,IC50)并按下述公式计算CR。
CR(%)=IC50(FB1)IC50(待检物)×100 式中:CR表示交叉反应率,%;IC50(FB1)表示FB1半数抑制浓度,μg/kg;IC50(待检物)表示待检物的半数抑制浓度,μg/kg。
1.2.7.3 检出限与定量限测试
取20个FB阴性玉米样本,按照1.2.5和1.2.6所述方法进行测试,并依据1.2.7.1所绘制标准曲线分别拟合FB1、FB2、FB3测量值,计算20份阴性样本平均值和标准差。检出限(Limits of detection,LOD)等于20份阴性样本读值的平均值加3倍标准差,定量限(Limits of quantitation,LOQ)等于20份阴性样读值的平均值加10倍标准差[27]。
1.2.7.4 准确度与精密度测试
在FB阴性玉米样本中分别添加浓度梯度为1000、2000、4000 μg/kg的FB1、FB2和FB3三类标准品。按照上述测试方法平行测试6次,汇总计算出准确度和精密度。其中准确度按回收率计算。
R(%)=AE×100 式中:R表示回收率,%;A表示测量值,μg/kg;E表示阳性样本的理论值,μg/kg。
精密度以变异系数计算。
CV(%)=AVRSTD×100 式中:CV表示变异系数,%;AVR表示6次测量结果平均值,μg/kg;STD表示6组平行测量结果的标准差,μg/kg。
1.2.7.5 与国标方法分析结果对比
用本研究所建立的时间分辨荧光免疫层析检测方法检测不同含量的FB实际阳性玉米样本,每个样本平行测试3次取平均值,与GB 5009.240-2016《食品安全国家标准食品中伏马毒素的测定》规定的第一法免疫亲和柱净化-柱后衍生高效液相色谱法[28]检测结果对比,通过符合度表述两种方法结果的一致性[19]。
符合度(%)=XY×100 式中:X表示TRFIA方法三次测量结果的平均值,μg/kg;Y表示IAC-HPLC方法测量值,μg/kg。
1.3 数据处理
拟合曲线时以添加的FB浓度值作为横坐标(每个浓度点测量6次取平均值),以时间分辨荧光免疫定量分析仪检测到的T线值之和与C线值的比值作为纵坐标用Origin 2018软件进行四参数拟合绘制曲线。包被条件确定时分别测试FB不同浓度点对应TC比值,测试三次取平均值,对比抑制率。确定检测限和定量限时阴性样本测量20次,计算平均值和标准差,进而计算出检测限和定量限。测试准确度时每个浓度点独立平行测试6次,计算均值与添加浓度对比,计算回收率。测试精密度时每个浓度点独立平行测试6次计算平均值和标准差,进一步计算出相对标准偏差。测试与国标方法结果相关性时,同一实际阳性样品独立平行测试3次取平均值,与国标方法测量结果进行符合度比较。
2. 结果与分析
2.1 FB膜包被模式确定
用制备的一条T线的FB膜和两条T线的FB膜,分别检测浓度为0、0.5、1、2、4、8 μg/kg的FB1标准品,测得各浓度点T/C和(T1+T2)/C值,每个浓度点测试三次取均值,通过公式得出各点抑制率数据(表1),两种模式下随标准品浓度变化对应显色变化如图3及图4。
包被模式 0 0.5 μg/kg 1 μg/kg 2 μg/kg 4 μg/kg 8 μg/kg 一条T线T/C值 5.79±0.09 4.34±0.01 3.45±0.11 2.71±0.02 1.72±0.02 1.03±0.04 两条T线(T1+T2)/C值 5.69±0.18 3.57±0.07 2.68±0.01 1.84±0.01 1.19±0.06 0.75±0.01 一条T线抑制率(%) 0 25.04 40.41 53.20 70.29 82.21 两条T线抑制率(%) 0 37.26 52.90 67.66 79.09 86.82 通过数据对比可知两条T线的FB膜在各个浓度点的抑制率均优于一条T线,表明两条T线的膜具有更好的灵敏度。导致此现象的原因是相对比两条T线模式,一条T线模式中抗原与抗体接触时间短,空间位阻大,抗原抗体反应不够充分,会导致灵敏度降低。在相关利用TRFIA方法检测其它霉菌毒素的研究中均证实了此结论[23,29−30]。因此本次研究将FB膜包被模式确定为两条T线的模式。
2.2 检测方法评价
2.2.1 定量曲线绘制
向阴性玉米样本中分别加入不同浓度梯度的FB1、FB2、FB3标准品,按已建立的前处理方法及检测方法进测试,每个浓度水平6组平行实验。以浓度值作为横坐标,以(T1+T2)/C的均值为纵坐标,用Origin 2018软件中logistic函数进行非线性拟合得到FB1、FB2、FB3随标准品浓度变化的定量曲线(图5)。荧光检测卡随标准品浓度变化对应显色变化如图6。
FB1拟合曲线的回归方程为:y=−0.58683+(4.88473+0.58683)/(1+(x/1897.5007)0.79534),决定系数R2=0.9987。FB2拟合曲线的回归方程为:y=−0.49451+(4.8806+0.49451)/(1+(x/2217.04761)0.77834),决定性系数R2=0.9988。FB3拟合曲线的回归方程为:y=−0.39709+(4.83214+0.39709)/(1+(x/2609.44216)0.92147),决定系数R2=0.9983。三种霉菌毒素的拟合曲线的决定系数均在0.99以上,表明曲线拟合良好。
2.2.2 特异性评价
分别拟合FB1、FB2、FB3、黄曲霉毒素B1、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、赭曲霉毒素A、T-2毒素的定量曲线,得出IC50值并以FB1为基准计算交叉反应率(表2)。结果显示与 FB2、FB3的交叉反应率分别为85.59%和72.72%,其余霉菌毒素在8000 μg/kg添加水平时仍无明显抑制,进行数据拟合计算IC50值,表明交叉反应率很低。所以本研究所建立的FB时间分辨荧光免疫层析检测方法FB1、FB2和FB3彼此交叉良好,可同时检测FB1、FB2、FB3。与其它霉菌毒素基本无交叉。检测方法对应的检测靶标是由所用抗体特异性决定的,本研究因选择对FB1、FB2和FB3交叉反应率更加全面又与其它真菌毒素交叉反应率低的FB抗体,从而保证了方法的特异性。
霉菌毒素类型 IC50(μg/kg) 交叉反应率(%) FB1 1897.50 100 FB2 2217.05 85.59 FB3 2609.44 72.72 2.2.3 检出限和定量限
采用上述所建立的时间分辨荧光免疫层析检测方法测量20份阴性样本,测得FB1的LOD为121.48 μg/kg,LOQ为316.48 μg/kg。FB2的LOD为107.68 μg/kg,LOQ为283.46 μg/kg。FB3的LOD为168.28 μg/kg,LOQ为444.63 μg/kg(表3),满足不同标准的限量要求[9−10]。
毒素类型 测量值(μg/kg,n=20) 平均值
(μg/kg)标准差
(μg/kg)检出限
(μg/kg)定量限
(μg/kg)FB1 0.00 48.87 0.00 10.11 5.92 51.52 73.55 65.12 0.00 59.62 37.90 27.86 121.48 316.48 21.38 43.64 23.74 51.52 3.92 85.10 43.64 28.55 59.62 82.18 FB2 0.00 41.19 0.00 6.82 3.65 43.72 65.33 56.96 0.00 51.55 32.35 25.11 107.68 283.46 16.12 36.24 18.16 43.72 2.24 76.99 36.24 22.40 74.02 51.55 FB3 0.00 64.01 0.00 8.75 3.58 67.99 101.67 88.68 0.00 80.26 49.85 39.48 168.28 444.63 23.94 56.19 27.26 67.99 1.35 119.62 56.19 34.10 115.07 80.26 2.2.4 准确度与精密度
采用上述所建立的时间分辨荧光免疫层析检测方法分别测量FB1、FB2、FB3含量为1000、2000、4000 μg/kg的加标样本,每个浓度点均做6次平行测试。FB1回收率在88.37%~114.20%之间,变异系数在5.44%~8.92%之间。FB2回收率在89.50%~111.51%之间,变异系数在3.99%~6.25%之间。FB3回收率在94.48%~117.42%之间,变异系数在3.94%~7.69%之间(表4)。优于伏马毒素胶体金定量检测标准方法中对准确度和精密度的要求[31]。
毒素类型 浓度(μg/kg) 6次检测结果(μg/kg) 均值(μg/kg) 回收范围(%) 标准差(μg/kg) 变异系数(%) FB1 1000 1116.73 1064.39 935.92 919.66 910.73 883.74 971.86 88.37~111.67 86.67 8.92 FB1 2000 2038.39 1947.80 1888.41 2284.08 2266.72 1934.60 2060.00 94.42~114.20 158.73 7.71 FB1 4000 3799.95 3929.37 4348.82 4071.72 4108.91 3684.30 3990.51 92.11~108.72 217.11 5.44 FB2 1000 1040.13 1077.32 1115.12 1027.54 931.92 955.52 1024.59 93.19~111.51 64.01 6.25 FB2 2000 1962.83 1940.59 1891.88 1789.99 2016.34 2034.76 1939.40 89.50~101.74 81.80 4.22 FB2 4000 3601.57 3701.57 3860.30 3710.68 4060.30 3901.44 3805.98 90.04~101.51 151.94 3.99 FB3 1000 1001.28 1000.29 1095.58 1174.25 1131.61 944.79 1057.97 94.48~117.42 81.35 7.69 FB3 2000 2279.83 2204.36 2085.59 2221.31 2326.19 2103.45 2203.46 104.28~116.31 86.71 3.94 FB3 4000 4489.94 4230.76 4294.26 4146.14 3803.43 4210.52 4195.84 95.09~112.25 205.78 4.90 2.2.5 与国标方法分析结果对比
用建立的时间分辨荧光免疫层析检测方法测定实际阳性玉米样本,每个样本平行测试3次取平均值,测量值与GB 5009.240-2016规定的IAC-HPLC方法的测量结果对比,计算符合度。结果符合度在92.17%~107.21%之间,表明两种方法检测同一样本时结果一致性良好(表5)。
毒素类型 IAC-HPLC
测量值(μg/kg)TRFIA测量值
(μg/kg,n=3)均值
(μg/kg)符合度
(%)FB1 1320 1360.45 1382.42 1250.20 1331.02 100.83 FB1 2530 2801.11 2653.34 2683.13 2712.53 107.21 FB1 3910 3500.39 3609.19 3702.31 3603.96 92.17 FB2 420 415.37 465.34 435.29 438.67 104.44 FB2 770 703.31 747.92 789.89 747.04 97.02 FB2 1140 1098.81 1273.31 1041.23 1137.78 99.81 FB3 490 490.12 510.34 509.18 503.21 102.69 3. 结论
本研究以铕元素时间分辨荧光微球作为示踪物,通过对比优化不同T线条数的划膜模式,最终建立伏马毒素时间分辨荧光侧向免疫层析检测方法。该方法可同时检测FB1、FB2和FB3,同时与其它常见真菌毒素无明显交叉,特异性良好,符合相关标准对伏马毒素的限量要求。方法的检出限为107.68~168.28 μg/kg,定量限为283.46~444.63 μg/kg,灵敏度良好。回收率在88.37%~117.42%之间,批内变异在3.94%~8.92%之间,准确度、精密度表现良好。此外该方法还具有操作简便、检测快速、成本低廉等优点,为FB的现场定量快速检测提供了可行的方法。
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表 1 不同T线条数抑制率对比
Table 1 Comparison of suppression rates for different numbers of T-lines
包被模式 0 0.5 μg/kg 1 μg/kg 2 μg/kg 4 μg/kg 8 μg/kg 一条T线T/C值 5.79±0.09 4.34±0.01 3.45±0.11 2.71±0.02 1.72±0.02 1.03±0.04 两条T线(T1+T2)/C值 5.69±0.18 3.57±0.07 2.68±0.01 1.84±0.01 1.19±0.06 0.75±0.01 一条T线抑制率(%) 0 25.04 40.41 53.20 70.29 82.21 两条T线抑制率(%) 0 37.26 52.90 67.66 79.09 86.82 表 2 所建方法与FB2、FB3及其它真菌毒素的交叉反应率
Table 2 Cross-reactivity of the established method with FB2, FB3 and other mycotoxins
霉菌毒素类型 IC50(μg/kg) 交叉反应率(%) FB1 1897.50 100 FB2 2217.05 85.59 FB3 2609.44 72.72 表 3 时间分辨荧光免疫层析方法的检出限与定量限
Table 3 Detection limit and quantification limit of the TRFIA method
毒素类型 测量值(μg/kg,n=20) 平均值
(μg/kg)标准差
(μg/kg)检出限
(μg/kg)定量限
(μg/kg)FB1 0.00 48.87 0.00 10.11 5.92 51.52 73.55 65.12 0.00 59.62 37.90 27.86 121.48 316.48 21.38 43.64 23.74 51.52 3.92 85.10 43.64 28.55 59.62 82.18 FB2 0.00 41.19 0.00 6.82 3.65 43.72 65.33 56.96 0.00 51.55 32.35 25.11 107.68 283.46 16.12 36.24 18.16 43.72 2.24 76.99 36.24 22.40 74.02 51.55 FB3 0.00 64.01 0.00 8.75 3.58 67.99 101.67 88.68 0.00 80.26 49.85 39.48 168.28 444.63 23.94 56.19 27.26 67.99 1.35 119.62 56.19 34.10 115.07 80.26 表 4 时间分辨荧光免疫层析方法的标准差和变异系数
Table 4 Standard deviation and coefficient of variation for TRFIA method
毒素类型 浓度(μg/kg) 6次检测结果(μg/kg) 均值(μg/kg) 回收范围(%) 标准差(μg/kg) 变异系数(%) FB1 1000 1116.73 1064.39 935.92 919.66 910.73 883.74 971.86 88.37~111.67 86.67 8.92 FB1 2000 2038.39 1947.80 1888.41 2284.08 2266.72 1934.60 2060.00 94.42~114.20 158.73 7.71 FB1 4000 3799.95 3929.37 4348.82 4071.72 4108.91 3684.30 3990.51 92.11~108.72 217.11 5.44 FB2 1000 1040.13 1077.32 1115.12 1027.54 931.92 955.52 1024.59 93.19~111.51 64.01 6.25 FB2 2000 1962.83 1940.59 1891.88 1789.99 2016.34 2034.76 1939.40 89.50~101.74 81.80 4.22 FB2 4000 3601.57 3701.57 3860.30 3710.68 4060.30 3901.44 3805.98 90.04~101.51 151.94 3.99 FB3 1000 1001.28 1000.29 1095.58 1174.25 1131.61 944.79 1057.97 94.48~117.42 81.35 7.69 FB3 2000 2279.83 2204.36 2085.59 2221.31 2326.19 2103.45 2203.46 104.28~116.31 86.71 3.94 FB3 4000 4489.94 4230.76 4294.26 4146.14 3803.43 4210.52 4195.84 95.09~112.25 205.78 4.90 表 5 时间分辨荧光免疫层析方法与国标仪器分析方法结果一致性
Table 5 Consistency between TRFIA method and national standard instrumental analysis method
毒素类型 IAC-HPLC
测量值(μg/kg)TRFIA测量值
(μg/kg,n=3)均值
(μg/kg)符合度
(%)FB1 1320 1360.45 1382.42 1250.20 1331.02 100.83 FB1 2530 2801.11 2653.34 2683.13 2712.53 107.21 FB1 3910 3500.39 3609.19 3702.31 3603.96 92.17 FB2 420 415.37 465.34 435.29 438.67 104.44 FB2 770 703.31 747.92 789.89 747.04 97.02 FB2 1140 1098.81 1273.31 1041.23 1137.78 99.81 FB3 490 490.12 510.34 509.18 503.21 102.69 -
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