荧光猝灭法测定梨小食心虫性诱剂含量以及田间应用

doi:10.16409/j.cnki.2095-039x.2021.03.032

摘要/Abstract

摘要： β-环糊精（β-CD）与中性红（NR）在碱性缓冲溶液中形成包合物（β-CD-NR），使中性红的荧光强度F增大，而加入梨小食心虫性诱剂主要成分顺-8-十二碳烯醇乙酸酯（Z8-12：Ac）后，β-CD-NR的荧光发生猝灭，以此建立了测定Z8-12：Ac含量的新方法：在含有1.0 mL 1.0×10^-3 mol/L中性红、5.0 mL 1.0×10^-2 mol/L的β-CD、7.0 mL 0.2 mol/L的NaH₂PO₄-Na₂HPO₄缓冲溶液（pH 7.4）的荧光体系中，加入Z8-12：Ac，以双蒸水定容至25.0 mL，超声反应20 min后，将其放置在4℃的环境下冷却，得待测液。在激发波长为460 nm、发射波长为580 nm的条件下测定待测液的荧光强度F和β-CD-NR体系的荧光强度F₀，计算荧光猝灭值ΔF（ΔF＝F₀—F）。比较分析不同浓度的Z8-12：Ac标准溶液与相应的荧光猝灭值ΔF的关系，结果表明：在Z8-12：Ac浓度为1.0×10^-4～4.0×10^-4 mol/L范围内，荧光猝灭值ΔF与Z8-12：Ac的浓度c呈现良好的线性关系，线性方程为：ΔF＝28.3 c—16.8，R²＝0.9978。对2.0×10^-4 mol/L的Z8-12：Ac溶液进行11次平行测定，得出方法检出限为1.25×10^-5 mol/L（S/N＝3），RSD为±1.5%，方法灵敏度和精密度良好。加标回收率在98.0%～103.0%，说明方法准确度良好。利用新方法测定了田间梨小诱芯中Z8-12：Ac随时间的残留量，统计了不同残留量对应的诱虫量。结果表明，本方法可以成功测定不同天数下诱芯中Z8-12：Ac的残留量，掌握了诱芯中Z8-12：Ac的衰减趋势，而且与实际诱虫量变化趋势基本吻合。本方法可为确保田间虫情监测预报的精确性以及保持性诱芯的诱杀效果提供技术支撑。

关键词: 梨小食心虫, 性诱剂, 荧光猝灭, 含量测定, 田间监测

Abstract: The aim of this research was to build a new way to determination of cis-8-dodecenyl acetate (Z8-12:Ac) content by fluorescence quench. In alkaline medium, β-cyclodextrin (β-CD) and neutral red (NR) formed an inclusion complex (β-CD-NR) to enhance the fluorescence intensity of neutral red (NR). However, the fluorescence of β-CD-NR system undergoes quench after adding Z8-12:Ac, the main component of sex attractant of Grapholitha molesta Busck. The solutions, which including NaH₂PO₄-Na₂HPO₄ buffer solution (pH 7.4, 0.2 mol/L,7.0 mL), neutral red solution (1.0×10^-3 mol/L, 1.0 mL) and β-CD solution (1.0×10^-2 mol/L, 5.0 mL), were pipette into 25.0 mL volumetric flask, mix the solution thoroughly and bring to volume after adding a certain amount of Z8-12:Ac. The mixture solution was ultrasonic reaction 20 minutes in an ultrasonic cleaner and kept at 4℃. The fluorescence intensity F of the above solution and the fluorescence intensity F₀ of the β-CD-NR system were analyzed by Fluorospectro Photometer (RF5301PC, JPN) under the conditions of excitation wavelength of 460 nm and emission wavelength of 580 nm. The fluorescence quenching value ΔF was calculated by the quotation:ΔF=F₀-F. It was compared and analyzed that the relationship between different concentrations of Z8-12:Ac standard solutions and the corresponding fluorescence quenching value ΔF. The results showed that the fluorescence quenching value ΔF showed a good linear relationship with the concentration of Z8-12:Ac in the range of 1.0×10^-4-4.0×10^-4 mol/L. The linear equation is ΔF=28.3c-16.8, R²=0.9978. ΔF value of 2.0×10^-4 mol/L Z8-12:Ac solution was measured parallelly 11 times to determine the method detection limits. The method detection limits was 1.25×10^-5 mol/L (S/N=3), RSD=±1.5%, indicated that the method has good precision. The spiked recoveries ranged from 98.0% to 103.0%, the method has good accuracy. This new method was used to test the concentrations of Z8-12:Ac in field experiments from lures and the amount of insects trapped corresponding to different residues was counted. This study elucidates that this method successfully determined the residual amount of Z8-12:Ac in the trap cores at different days and knew the decay trend of Z8-12:Ac in the trap cores. Its attenuation trend is basically consistent with the change trend of insect trapping quantity in the field. This method can provide technical support for the field monitoring of G. molesta trap cores, the accuracy of insect forecasting, and ensure the trapping effect of the cores.

Key words: Grapholitha molesta, sex pheromone, fluorescence quenching, content determination, field monitoring

中图分类号:

S476

窦蕊, 杜少芳, 王洋, 荆小院, 马瑞燕, 刘金龙. 荧光猝灭法测定梨小食心虫性诱剂含量以及田间应用[J]. 中国生物防治学报, 2021, 37(2): 385-393.

DOU Rui, DU Shaofang, WANG Yang, JING Xiaoyuan, MA Ruiyan, LIU Jinlong. Fluorescence Quenching Method for Determining the Content of Sexual Attractants of Grapholitha molesta Busck and Its Field Application[J]. Chinese Journal of Biological Control, 2021, 37(2): 385-393.

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