交链孢酚适配体荧光试纸条检测方法的建立及应用

doi:10.19586/j.2095-2341.2024.0144

生物技术进展 ›› 2024, Vol. 14 ›› Issue (6): 1024-1031.DOI: 10.19586/j.2095-2341.2024.0144

交链孢酚适配体荧光试纸条检测方法的建立及应用

方梦旗¹^,²(), 赵颖¹, 王子晨¹^,², 贾文昊², 王辉²(), 栾云霞¹()

^1.北京市农林科学院质量标准与检测技术研究所，农业农村部农产品质量安全风险评估实验室（北京），北京 100097
^2.北京城市学院生物医药学部，北京 100191

收稿日期:2024-09-01 接受日期:2024-09-27 出版日期:2024-11-25 发布日期:2024-12-27
通讯作者: 王辉,栾云霞
作者简介:方梦旗 E-mail: llie_fmq@163.com
基金资助:
北京市农林科学院科技创新专项建设资助项目(KJCX20230437)

Establishment and Application of Aptamer-based Fluorescent Test Strip Method for the Detection of Alternariol

Mengqi FANG¹^,²(), Ying ZHAO¹, Zichen WANG¹^,², Wenhao JIA², Hui WANG²(), Yunxia LUAN¹()

^1.Agricultural Product Quality and Safety Risk Assessment Laboratory （Beijing） of the Ministry of Agriculture and Rural Affairs，Institute of Quality Standard and Testing Technology，Beijing Academy of Agriculture and Forestry Sciences，Beijing 100097，China
^2.Biomedical Department of Beijing City University，Beijing 100191，China

Received:2024-09-01 Accepted:2024-09-27 Online:2024-11-25 Published:2024-12-27
Contact: Hui WANG,Yunxia LUAN

摘要/Abstract

摘要：

交链孢酚（alternariol，AOH），作为交链孢霉菌产生的一种有毒代谢产物，通过侵染小麦、番茄、樱桃和柑橘等农产品，不仅造成经济损失，还对人类健康构成威胁。基于竞争原理，构建了一种针对AOH的灵敏且特异性强的核酸适配体荧光试纸条检测体系。对适配体浓度、pH和缓冲液等条件进行优化后，进一步评价其准确性和特异性等性能，并应用于小麦样品中AOH的含量测定。在最优条件下，其检测限可达1 μg·L^-1，检测范围为1~1 000 μg·L^-1，线性相关系数R²=0.979 2，仅需20 min即可完成检测。在实际样品分析中的回收率为96.6%~114.3%，相对标准偏差为2.6%~9.0%。利用核酸适配体作为识别元件，开发了一种简便、快速、准确的适合现场快速定量检测AOH的新方法，为其他真菌毒素快速检测技术的研发提供了参考。

关键词: 核酸适配体, 荧光侧流层析试纸条, 交链孢酚, 小麦

Abstract:

Alternariol （AOH） is a toxic metabolite produced by the fungus Alternariol. By infecting wheat， tomato， cherry， citrus and other agricultural products， Streptomyces alternans not only causes economic losses but also endangers human health. In this study， based on the principle of competition， a sensitive and specific aptamer fluorescence test strip for AOH detection was constructed. After optimizing the aptamer concentration， pH value and buffer， the accuracy and specificity of the aptamer were further evaluated and applied to the determination of AOH in wheat samples. Under the optimal conditions， the detection limit was 1 μg·L^-1， the detection range was 1~1 000 μg·L^-1， the linear correlation coefficient R²=0.979 2， the detection could be completed in only 20 minutes， the recovery rate in the actual sample analysis was 96.6%~114.3%， the relative standard deviation was 2.6%~9.0%. In this study， a simple， rapid and accurate new method for rapid quantitative detection of AOH was developed using aptamer as a recognition element， which would provide a reference for the development of other mycotoxin rapid detection technologies.

Key words: aptamer, fluorescent lateral flow test strips, alternariol, wheat

中图分类号:

Q524

方梦旗, 赵颖, 王子晨, 贾文昊, 王辉, 栾云霞. 交链孢酚适配体荧光试纸条检测方法的建立及应用[J]. 生物技术进展, 2024, 14(6): 1024-1031.

Mengqi FANG, Ying ZHAO, Zichen WANG, Wenhao JIA, Hui WANG, Yunxia LUAN. Establishment and Application of Aptamer-based Fluorescent Test Strip Method for the Detection of Alternariol[J]. Current Biotechnology, 2024, 14(6): 1024-1031.

图/表 8

表1 适配体和互补链序列的设计

Table 1 Design of aptamer and complementary strand sequences

序列名称	修饰的序列（5' $→$ -3'）
Aptamer	5'-Cy5-AAA AAA AAA AAA AAA AAA AGG GAC GGG ATA GCT TAA CTA GTG TTC AAG CTG AGG GAC GGG A-3'
Probe 1	5'-Biotin-TCC CGT CCC TCA GCT TGA ACA CTA GTT AAG CTA TCC CGT CCC T-3'
Probe 2	5'-Biotin-TTT TTT TTT TTT TTT TTT-3'

表1 适配体和互补链序列的设计

Table 1 Design of aptamer and complementary strand sequences

序列名称	修饰的序列（5' $→$ -3'）
Aptamer	5'-Cy5-AAA AAA AAA AAA AAA AAA AGG GAC GGG ATA GCT TAA CTA GTG TTC AAG CTG AGG GAC GGG A-3'
Probe 1	5'-Biotin-TCC CGT CCC TCA GCT TGA ACA CTA GTT AAG CTA TCC CGT CCC T-3'
Probe 2	5'-Biotin-TTT TTT TTT TTT TTT TTT-3'

图 1 荧光适配体试纸条技术检测AOH的示意图

Fig. 1 Schematic diagram of the fluorescent aptamer test strip technique for the detection of AOH

图 2 适配体浓度的优化（n=3）A：T/C与(T/C)'的荧光比值；B：T/C与(T/C)'的荧光比值的差值

Fig. 2 Optimisation of aptamer concentration (n=3)

图 3 最佳缓冲液的确定（n=3）A：T/C与(T/C)'的荧光比值；B：T/C与(T/C)'的荧光比值的差值

Fig. 3 Optimisation of buffers (n=3)

图 4 荧光适配体试纸条法检测AOH（n=3）A：荧光光谱图；B：线性关系图

Fig. 4 Detection of AOH by fluorescent aptamer test strip method (n=3)

图 5 适配体试纸条检测交链孢酚的特异性

Fig. 5 Specificity of aptamer test strips

图 6 小麦中检测AOH的线性关系

Fig. 6 Linearity of AOH detection in wheat

表2 小麦样品中加标回收率的测定（n=3）

Table 2 Determination of spiked recoveries in wheat samples (n=3)

加标量/（μg·L^-1）	检出量/（μg·L^-1）	回收率/%	RSD/%
50	57.17	114.3	9.0
200	204.02	102.0	5.3
800	772.96	96.6	2.6

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交链孢酚适配体荧光试纸条检测方法的建立及应用

Establishment and Application of Aptamer-based Fluorescent Test Strip Method for the Detection of Alternariol

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