农产品中黄曲霉毒素产毒菌标识性分子大容量反应体系 提高ELISA灵敏度

doi:10.3864/j.issn.0578-1752.2020.07.015

中国农业科学 ›› 2020, Vol. 53 ›› Issue (7): 1473-1481.doi: 10.3864/j.issn.0578-1752.2020.07.015

农产品中黄曲霉毒素产毒菌标识性分子大容量反应体系提高ELISA灵敏度

魏晓^1,^2,⁴,张奇^1,^2,³,张文^1,^3,⁵,李慧^1,^2,⁴,李培武^1,^2,^3,^4,⁵

1．中国农业科学院油料作物研究所,武汉 430062
^2. 农业部油料作物生物学与遗传育种重点实验室,武汉430062
^3. 农业部生物毒素检测重点实验室,武汉 430062
^4. 农业部油料产品质量安全风险评估实验室,武汉 430062
^5. 农业部油料及制品质量监督检验测试中心,武汉 430062

收稿日期:2019-08-09 接受日期:2019-11-05 出版日期:2020-04-01 发布日期:2020-04-14
作者简介:魏晓,Tel：13163292893;E-mail：15621567212@163.com。
基金资助:
国家重点研发计划(2018YFC1602505);国家自然科学基金(31801665);湖北省技术创新专项重大项目(2018ABA081);湖北省自然科学基金(2017CFB333)

Improving the Sensitivity of ELISA by Large-Capacity Reaction System of Aflatoxigenic Fungi-Biomarker in Agro-products

Xiao WEI^1,^2,⁴,Qi ZHANG^1,^2,³,Wen ZHANG^1,^3,⁵,Hui LI^1,^2,⁴,PeiWu LI^1,^2,^3,^4,⁵

^1. Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062
^2. Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062
^3. Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan 430062
^4. Laboratory of Risk Assessment for Oilseeds Products (Wuhan) , Ministry of Agriculture, Wuhan 430062
^5. Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan 430062

Received:2019-08-09 Accepted:2019-11-05 Online:2020-04-01 Published:2020-04-14

摘要/Abstract

摘要： 【目的】 为预防和降低黄曲霉毒素污染,在前期探明黄曲霉毒素产毒菌株鉴别标识性分子PO8蛋白的基础上,欲研究建立高灵敏、大容量反应体系的双抗夹心ELISA检测技术,为污染源头监控提供技术支撑。 【方法】 用干菌丝作标识性分子PO8蛋白的参考物,以高压均质制得的黄曲霉菌裂解液为检测抗原,纯化后的PO8-VHH作包被抗体,产毒菌株多抗作检测抗体,抗原、抗体分别以200 μL/孔加入到96孔酶标板,进行大容量反应体系夹心ELISA法的试验。优化相关理化因素,以阳性孔OD_450nm≥1.0,阳性孔OD_450nm/阴性孔OD_450nm较高为原则,确定最佳试验条件,建立标准曲线。并通过样品添加回收试验、重复性试验、特异性试验,对建立的夹心ELISA方法进行性能评估。 【结果】 通过棋盘格试验确定了最佳包被抗体浓度为3.0 μg?mL ^-1,最佳多抗的工作浓度为2.5 μg?mL ^-1。优化反应条件确定：最佳抗体包被条件为4℃过夜,最佳封闭液为3%的BSA,最佳封闭条件为37℃封闭2 h,最佳多抗作用条件为37℃反应50 min。在优化后的条件下建立了夹心ELISA方法的标准曲线,对黄曲霉菌检测限可达到0.1 μg?mL ^-1,比前期报道的常规容量反应体系灵敏度提高了约10倍。该方法特异性强,与青霉菌、尖孢镰刀菌、串珠镰刀菌、赭曲霉菌均无交叉反应,且检测非产毒黄曲霉菌株信号值较低,接近于阴性值。重复性试验显示,板间变异系数为1.5%—5.8%,板内变异系数为0.4%—3.2%,均小于7%,说明该方法稳定性好。采用该方法对花生、玉米等农产品进行添加回收试验,平均回收率在81.9%—109.0%。 【结论】 本研究建立的大容量反应体系夹心ELISA方法可快速、高灵敏地检测黄曲霉毒素产毒菌,为从源头上控制黄曲霉毒素污染提供了新的快速、方便的检测技术支撑。

关键词: 农产品, 黄曲霉菌, 纳米抗体, ELISA, 标识性分子

Abstract: 【Objective】In order to prevent and reduce the contamination by Aspergillus species from the source, a highly sensitive large-capacity reaction system DAS-ELISA was established, based on the biomarker PO8 protein of aflatoxigenic fungi. This study aimed to provide key technical support for pollution source monitoring. 【Method】 In this study, the dry mycelium was used as a reference for the biomarker PO8 protein, the mycelia lysate made by high pressure homogenization was used as envelope antigen, the purified PO8-VHH was used as capture antibody, and rabbit polyclonal antibody against Aspergillus was used as detection antibody. The antigen and antibody were added to the 96-well microtiter plate at 200 μL/well, and the sandwich ELISA for the large-capacity reaction system was carried out. Based on the principle that the positive hole OD_450nm≥1.0, the positive hole OD_450nm/negative hole OD_450nm was higher to determine the optimal experimental conditions and to establish a standard curve. The performance of the established sandwich ELISA method was evaluated by spike-and-recovery test, repeatability test and specific test. 【Result】 Assays were performed in the PO8-VHH (3 μg?mL ^-1) coated ELISA format, in which the detection antibody was 2.5 μg?mL ^-1 diluted. The optimized physicochemical factors in the performance were obtained: the antibody coating condition was 4℃ overnight, the blocking reagent was 3% BSA, the blocking condition was 37℃ 2 h, and the polyclonal antibody working time was 50 min. The standard curve was established under the optimal conditions, the minimum detectable limit was 0.1 μg?mL ^-1. This method was specific, with no cross reaction with HBHA, FO, FV, and AO. Meanwhile, the inter-assay repetition rate was 1.5%-5.8% and intra-assay repetition rate was 0.4%-3.2%, both lower than 10%, indicating it was good repeatability. Non-aflatoxigenic fungi had lower values, close to negative value. 【Conclusion】 The large-capacity reaction system sandwich ELISA method established in this study could quickly and accurately detect aflatoxigenic fungi, which laid a foundation for further control of aflatoxin contamination from the source.

Key words: agro-products, Aspergillus flavus, nanobody, ELISA, biomarker

魏晓,张奇,张文,李慧,李培武. 农产品中黄曲霉毒素产毒菌标识性分子大容量反应体系提高ELISA灵敏度[J]. 中国农业科学, 2020, 53(7): 1473-1481.

Xiao WEI,Qi ZHANG,Wen ZHANG,Hui LI,PeiWu LI. Improving the Sensitivity of ELISA by Large-Capacity Reaction System of Aflatoxigenic Fungi-Biomarker in Agro-products[J]. Scientia Agricultura Sinica, 2020, 53(7): 1473-1481.

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包被抗体稀释度 Antibody coating dilution (μg∙mL^-1)	多抗稀释度 Polyclonal antibody concentration (μg∙mL^-1)
包被抗体稀释度 Antibody coating dilution (μg∙mL^-1)	4	3	2.5	2	1.5	1	0.5
0.5	1.4758	0.8651	0.5182	0.3603	0.3344	0.227	0.2148
1	1.5511	0.8667	0.5633	0.4631	0.3676	0.3353	0.2937
2	1.5714	0.9132	0.6063	0.4855	0.3974	0.3598	0.2949
3	1.8485	1.3707	1.0641	0.7815	0.5402	0.3655	0.3570

项目 Project	检测样品 Test sample
项目 Project	阳性 Positive	阴性 Negative	HBHA	FO	FV	AO
OD_450nm	1.043	0.179	0.172	0.221	0.176	0.169

组别 Sample number	OD_450nm值 OD_450nmvalue	AFB₁的含量 Concentration of AFB₁(μg∙kg^-1)
1	0.201	0.000
2	0.202	0.000
3	0.209	0.000
4	0.199	0.000
5	0.193	0.000
6	0.389	1237.053
7	0.417	2294.187
8	0.388	3135.416
9	0.470	4212.220
10	0.466	5434.766

组别 Sample number	平均值 Average value	标准差 Standard deviation	变异系数 Variation coefficient (%)
阳性1 Positive1	1.975	0.033	1.6
阳性2 Positive2	1.043	0.047	4.5
阳性3 Positive3	0.581	0.015	2.6
阳性4 Positive4	0.523	0.008	1.5
阴性5 Negative5	0.188	0.011	5.8

组别 Sample number	平均值 Average value	标准差 Standard deviation	变异系数 Variation coefficient (%)
阳性1 Positive1	1.725	0.006	0.4
阳性2 Positive2	1.069	0.012	1.2
阳性3 Positive3	0.585	0.019	3.2
阳性4 Positive4	0.532	0.014	2.6
阴性5 Negative5	0.184	0.004	2.0

农产品中黄曲霉毒素产毒菌标识性分子大容量反应体系提高ELISA灵敏度

Improving the Sensitivity of ELISA by Large-Capacity Reaction System of Aflatoxigenic Fungi-Biomarker in Agro-products

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参考文献 41

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样品 Sample	添加量 Dosage (μg∙mL^-1)	检出浓度 Detection concentration (μg∙mL^-1)	回收率 Rate of recovery (%)
花生 Peanut	100	107.32±3.20	107.32
	10	8.19±1.08	81.90
	1	0.97±0.12	97.00
玉米 Corn	100	105.55±3.80	105.55
	10	9.6±0.98	96.00
	1	1.09±0.13	109.00

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农产品中黄曲霉毒素产毒菌标识性分子大容量反应体系 提高ELISA灵敏度

Improving the Sensitivity of ELISA by Large-Capacity Reaction System of Aflatoxigenic Fungi-Biomarker in Agro-products

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农产品中黄曲霉毒素产毒菌标识性分子大容量反应体系提高ELISA灵敏度