苹果褪绿叶斑病毒RT-LAMP检测方法的建立

doi:10.3864/j.issn.0578-1752.2018.09.008

摘要/Abstract

摘要： 【目的】建立一种利用反转录环介导等温扩增（reverse transcription loop-mediated isothermal amplification，RT-LAMP）技术简便、快速检测苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus, ACLSV）的方法。【方法】在ACLSV基因组序列的3个保守区域设计3组引物，每组引物包括一对外引物（F3/B3）和一对内引物（FIP/BIP），从3组引物中筛选出一组效果最好的引物。对RT-LAMP反应体系，即Mg²⁺、dNTPs、Betaine、FIP/BIP、B3/F3浓度进行优化，Mg²⁺浓度梯度设置为0、2.0、4.0、6.0、8.0、10.0、12.0 mmol·L^-1，dNTPs浓度为0.6、0.8、1.0、1.2、1.4、1.6、1.8 mmol·L^-1，Betaine浓度为0、0.2、0.4、0.6、0.8、1.0、1.2 mol·L^-1，FIP/BIP浓度为0.8、1.2、1.6、2.0、2.4 µmol·L^-1，F3/B3浓度为0、0.1、0.2、0.3、0.4 µmol·L^-1；优化RT-LAMP反应条件，采用已优化的反应体系，设置65、63、61、59、57℃ 5个不同的反应温度，反应时间设定为90 min。在引物筛选和反应体系反应条件优化过程中，使用荧光定量PCR仪，在反应体系中加入荧光染料，利用荧光信号积累实时监测整个反应过程，根据扩增曲线判断反应结果。以携带苹果茎沟病毒(Apple stem grooving virus，ASGV)、苹果茎痘病毒(Apple stem pitting virus，ASPV)、苹果花叶病毒 (Apple mosaic virus , ApMV)的植株叶片中提取的总RNA为模板测试RT-LAMP检测方法的特异性。将含ACLSV的叶片总RNA原液进行10倍梯度稀释，以RNA原液和10^-1、10^-2、10^-3、10^-4、10^-5、10^-6稀释液作为模板，进行RT-LAMP反应，测试RT-LAMP检测方法的灵敏性。随机采集23株苹果树的叶片，同时进行RT-LAMP和RT-PCR检测，加入SYBR GreenⅠ进行可视化检测。【结果】建立了ACLSV RT-LAMP检测方法，优化的检测体系为：6.0 mmol·L^-1 Mg²⁺、1.2 mmol·L^-1 dNTPs、0.2 mol·L^-1 Betaine、1.6 µmol·L^-1 FIP/BIP和0.2 µmol·L^-1 F3/B3引物，最佳反应条件为59℃，60 min。特异性检测中，仅ACLSV检测结果为阳性，对照组均为阴性。灵敏性检测中，RT-LAMP方法最低可检测到10^-3 RNA稀释液,灵敏度是RT-PCR方法的100倍。随机采取的23株苹果叶片样品RT-PCR阳性检出率为52.2%，RT-LAMP阳性检出率为65.2%，RT-LAMP的检出率高于RT-PCR。【结论】建立的ACLSV RT-LAMP检测方法具有简便、快速、灵敏性高、成本低等特点，可满足基层、科研部门在田间调查、种苗繁育和海关检疫中快速检测ACLSV。

关键词: 苹果；苹果褪绿叶斑病毒；反转录环介导等温扩增；real-time PCR, 病毒检测

Abstract: 【Objective】The objective of this study is to establish a method which uses reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology, and to detect Apple chlorotic leaf spot virus (ACLSV) simply and quickly. 【Method】Three sets of specific primers were designed based on conserved region of ACLSV genomes. Each set of primers includes a pair of outer primer (F3/B3) and a pair of inner primer (FIP/BIP). One feasible set of primers was selected for the RT-LAMP reaction. RT-LAMP reaction system was optimized, that is, the concentration of Mg²⁺ (0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 mmol·L^-1), dNTPs (0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 mmol·L^-1), Betaine (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mol·L^-1), FIP/BIP (0.8, 1.2, 1.6, 2.0, 2.4 µmol·L^-1) and F3/ B3 (0, 0.1, 0.2, 0.3, 0.4 µmol·L^-1), respectively. The RT-LAMP reaction condition was optimized, using optimized reaction system, reaction temperature was set to 65, 63, 61, 59, 57℃ and reaction time was set for 90 min. In the process of primers screening and optimizing the reaction system, fluorescent pigment was added to the reaction system and real-time PCR instrument was used. The whole process was detected in real-time by fluorescence signal accumulation. Finally, the amplification curve was used to analyze the reaction result. the specificity detection of RT-LAMP was tested by using different RNA templates of infected leaves from ACLSV, Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple mosaic virus (ApMV). To assess the detection sensitivity, 10⁰, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6diluents of original RNA were used as templates of RT-LAMP. To evaluate the application value of this method, 23 apple leaf samples were collected randomly in the field, the optimized RT-LAMP and RT-PCR were used to detect the samples, SYBR GreenⅠwas added to visualize the detection. 【Result】 The RT-LAMP method to detect ACLSV was established. The optimized detection system was 6.0 mmol·L^-1 Mg²⁺, 1.2 mmol·L^-1 dNTPs, 0.2 mol·L^-1 Betaine, 1.6 µmol·L^-1 FIP/BIP and 0.2 µmol·L^-1 F3/B3 primer. The optimum reaction conditions were 59℃ and 60 min. In the specificity test, only the ACLSV test result was positive, the controls were all negative. In the sensitivity test, the 10^-3 RNA dilution could be detected by RT-LAMP method. It was 100 times higher sensitivity than RT-PCR method. The positive rate of RT-PCR of randomly selected 23 apple leaf samples was 52.2%, and RT-LAMP was 65.2%. Detection rate of RT-LAMP was higher than that of RT-PCR. 【Conclusion】The established ACLSV RT-LAMP detection method is simple, quick, sensitive and low cost. It can be applied in field investigation, seeding breeding and customs quarantine.

Key words: apple, Apple chlorotic leaf spot virus (ACLSV), reverse transcription loop-mediated isothermal amplification (RT-LAMP), real-time PCR, virus detection

张双纳，李正男，范旭东，张尊平，任芳，胡国君，董雅凤. 苹果褪绿叶斑病毒RT-LAMP检测方法的建立[J]. 中国农业科学, 2018, 51(9): 1706-1716.

ZHANG ShuangNa, LI ZhengNan, FAN XuDong, ZHANG ZunPing, REN Fang, HU GuoJun, DONG YaFeng. Establishment of RT-LAMP Assay for Detection of Apple chlorotic leaf spot virus (ACLSV)[J]. Scientia Agricultura Sinica, 2018, 51(9): 1706-1716.

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