环介导等温扩增（LAMP）技术检测蜜蜂球囊菌

doi:10.3864/j.issn.0578-1752.2016.04.015

摘要/Abstract

摘要： 【目的】建立一种快速、灵敏的检测蜜蜂球囊菌（Ascosphaera apis）的环介导等温扩增(loop-mediated isothermal amplification，LAMP)方法，为监测和防治蜜蜂白垩病提供技术支撑。【方法】根据蜜蜂球囊菌特异性序列ITS区，用在线引物设计软件PrimerExplorer V4.0设计并合成4条特异性引物A.apis-F3 (5′-ACATTGCGCCCTCTGGTA-3′)、A.apis-B3 (5′-TGGTTAGACCGGACAGTCG-3′)、A.apis-FIP (5′-TAAGACGGGACGATCGCCC AACCTGTCCGAGCGTCATTG-3′)和 A.apis-BIP (5′-GAAAGGCAGTGACGGCGTCGGGCCACTAGAGCGAAAGAC-3′)，进行LAMP扩增试验，分别设置Mg²⁺终浓度为0、2、4、6、8、10、12、14 mmol·L^-1, dNPTs终浓度为0、0.2、0.4、0.6、0.8、1.0、1.2、1.4、1.6、1.8 mmol·L^-1, 内引物FIB/BIF终浓度为0.2、0.4、0.6、0.8、1.0、1.2、1.4、1.6、1.8 mmol?L^-1，甜菜碱终浓度分别为0、0.2、0.4、0.6、0.8、1.0、1.2 mol·L^-1，反应温度为58、60、63、65℃，反应时间分别为30、40、50、60 min，并利用实时浊度仪测定浊度值和扩增产物进行琼脂糖凝胶电泳来检测LAMP反应的结果，确定优化的LAMP检测体系。以东方蜜蜂微孢子虫（Nosema ceranae）、蜜蜂微孢子虫（Nosema apis）、蜜蜂残翅病毒（Deformed wing virus，DWV）、蜜蜂囊状幼虫病毒（Sacbrood virus，SBV）、黑蜂王台病毒（Black queen cell virus，BQCV）和以色列急性麻痹病毒（Israel acute paralysis virus，IAPV）基因组为模板进行特异性验证。并用PvuⅠ酶切验证产物，最终确定LAMP反应体系的准确性；进而通过将蜜蜂球囊菌基因组DNA进行10倍梯度稀释，分别获得DNA浓度0.2231、0.2231×10^-1、0.2231×10^-2、0.2231×10^-3、0.2231×10^-4、02231×10^-5、0.2231×10^-6、0.2231×10^-7、0.2231×10^-8 μg·μL^-1作为模板，比较LAMP和普通PCR检测灵敏度，并检测验证该技术在临床应用上的可行性。【结果】建立了一种特异检测蜜蜂球囊菌的方法，反应条件优化后的检测体系为4 mmol·L^-1Mg²⁺、1.2 mmol·L^-1dNTPs、1.6 mmol·L^-1FIP/BIP、0.4 mol·L^-1甜菜碱，并在63℃反应60 min可完成检测。选用蜜蜂球囊菌、东方蜜蜂微孢子虫、蜜蜂微孢子虫、蜜蜂残翅病毒、蜜蜂囊状幼虫病毒、黑蜂王台病毒和以色列急性麻痹病毒的基因组作为LAMP反应模板进行特异性检测，结果显示仅有蜜蜂球囊菌有扩增曲线和梯状条带，建立的LAMP检测体系有很好的特异性。将蜜蜂球囊菌基因组DNA浓度0.2231、0.2231×10^-1、0.2231×10^-2、0.2231×10^-3、0.2231×10^-4、0.2231×10^-5μg·μL^-1作为模板进行PCR反应后，检测结果为阳性，随DNA浓度降低，电泳检测不能观察到PCR的扩增产物。进行LAMP反应时，浊度曲线和电泳条带显示可检测DNA浓度为0.2231×10^-6μg·μL^-1。LAMP每反应检出量比PCR高10倍，并且方法简单、节省时间。【结论】成功建立了准确、快速、低成本的LAMP检测蜜蜂球囊菌技术，为相关研究和应用提供了技术支持。

关键词: 环介导等温扩增, 蜜蜂球囊菌, 快速检测, 实时浊度

Abstract: 【Objective】 The objective of this study is to establish a rapid and sensitive method for detecting the Ascosphaera apis in epidemiological investigations by the loop-mediated isothermal amplification (LAMP), and to provide technical supports for monitoring and prevention of chalkbrood in honeybee.【Method】Four specific primers were designed based on the particular sequence of ITS of the A. apis using the software of PrimerExplorer V4.0 online. The primers included A. apis-F3 (5′-ACATTGC GCCCTCTGGTA-3′), A. apis-B3 (5′-TGGTTAGACCGGACAGTCG-3′), A. apis-FIP (5′-TAAGACGGGACGATCGCCCAACC TGTCCGAGCGTCATTG-3′) and A. apis-BIP (5′-GAAAGGCAGTGACGGCGTCGGGCCACTAGAGCGAAAGAC-3′). LAMP were performed under different concentrations such as Mg²⁺ (0, 2, 4, 6, 8, 10, 12, and 14 mmol·L^-1), dNPTs (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mmol?L^-1), FIB/BIF (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mmol·L^-1), betaine (0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mol?L^-1), reaction temperatures (58, 60, 63, and 65℃) and reaction times (30, 40, 50, and 60 min), respectively. The LAMP result was determined by both the turbidimeter and agarose gel electrophoresis in order to optimize the reaction conditions. The specificity of LAMP was tested by using genomic DNA of Nosema ceranae, Nosema apis, Deformed wing virus (DWV), Sacbrood virus (SBV), Black queen cell virus (BQCV) and Israel acute paralysis virus (IAPV). In addition, the LAMP amplified products were digested with PvuⅠrestriction enzyme. The sensitivity of LAMP was compared with normal PCR by employing ten-fold serially diluted DNA templates of A. apis (including 0.2231, 0.2231×10^-1, 0.2231×10^-2, 0.2231×10^-3, 0.2231×10^-4, 0.2231×10^-5, 0.2231×10^-6, 0. 2231×10^-7, and 0.2231×10^-8 μg·μL^-1). The practical reliability was achieved in clinical trials.【Result】A rapid and specific method for detction of A. apis was established. The optimum conditions for LAMP reaction were as follows: 4 mmol·L^-1 Mg²⁺, 1.2 mmol·L^-1dNTPs, 1.6 mmol·L^-1FIP/BIP, 0.4 mol·L^-1betaine, and the method was performed at 63℃ for 60 min. The templates of A. apis, N. ceranae, N. apis, DWV, SBV, BQCV and IAPV were tested, the result showed that only the A. apis DNA template could produce typical ladder-like bands. The sensitivity test showed that the PCR could detect the DNA templates as low as 0.2231×10^-5 μg·μL^-1. While the LAMP could detect ten times lower DNA templates of 0.2231×10^-6μg·μL^-1. The LAMP is effective, simple and time saving for detecting the A. apis infect honeybee. 【Conclusion】The method of LAMP was proved to be a precise, faster, lower cost in diagnosis of chalkbrood in honeybee. It also could be applied in both research institutions and field.

Key words: loop-mediated isothermal amplification (LAMP), Ascosphaera apis, rapidly detective, real-time turbidity

席伟军，李江红，陈大福，梁勤. 环介导等温扩增（LAMP）技术检测蜜蜂球囊菌[J]. 中国农业科学, 2016, 49(4): 765-774.

XI Wei-jun, LI Jiang-hong, CHEN Da-fu, LIANG Qin. Diagnosis of the Ascosphaera apis by the Loop-Mediated Isothermal Amplification[J]. Scientia Agricultura Sinica, 2016, 49(4): 765-774.

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