Establishment and Application of A Loop-mediated Isothermal Amplification Assay for Rapid Detection of Phellinus noxius on Hevea brasiliensis
DONG Wen-Min1,2,*, HE Chun-Ping2,*, WU Wei-Huai2, LIANG Yan-Qiong2, XIE Li3, LI Rui2, HUANG Xing2, ZHENG Jin-Long2, XI Jin-Gen2, LU Ying2, YI Ke-Xian1,2,**
1 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; 2 Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Integrated Pest Management on Tropical Grops, Ministry of Agriculture and Rural Affairs, China/Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Haikou 571101, China; 3 Institute of Forestry, Hainan University, Haikou 570228, China
Abstract：Rubber tree (Hevea brasiliensis) brown root disease caused by Phellinus noxius is a root disease that seriously harms rubber. Loop-mediated isothermal amplification (LAMP) has been widely used in many fields such as plant protection because of its high specificity, high sensitivity, rapid response, and low cost. Therefore, an acturate, sensitive and rapid detection method is a powerful tool to timely effective control measures. In this study, SYBR Green Ⅰ as an indicator, a fast visual detection method of LAMP for the detection of P. noxius was established. Four primers (2 inner primers FIP/BIP, 2 outer primers F3/B3) were designed for LAMP amplification based on the ribosome transcriptional spacer sequence specific to P. noxius. The reaction conditions were optimized and the specificity and sensitivity of LAMP were assayed. The suspected brown root disease samples in the field were verified. The study determined the optimal reaction system (100 ng/μL for template in 25 μL, 0.2 μmol/L for outer primer F3/B3, 1.2 μmol/L for inner primer FIP/BIP, 1.4 mmol/L for dNTPs, and 6 mmol/L for Mg2+, betaine is 0.8 mol/L, Bst DNA polymerase is 8 U/μL and optimal reaction temperature 63 °C, reaction 1 h). Specificity assays showed that a positive color (green) was only observed in the presence of P. noxius by SYBR GreenⅠas an indicator reaction prior to amplification, however none of other pathogens changed color (still orange). The lower limit of detection of the LAMP assay was about 1 pg/μL of genomic DNA, which was 1 000 times more sensitive than PCR method. Twenty strains of suspected samples in the field were tested by the LAMP system with the pathogen detection rate was 85%, while thenormal PCR detection rate was only 70% or 75%. The LAMP detection system established in this study can be used for the rapid detection of suspected samples of root rot of rubber tree, which was of great significance for effectively controlling the spread of the disease.
[1] 蔡怡, 周前进, 顾建锋, 等. 2016. 山茶根结线虫的LAMP-LFD快速检测方法[J]. 农业生物技术学报, 24(05): 770-780. (Cai Y, Zhou Q J, Gu J F, et al.2016. Rapid and sensitive detection of Meloidogyne camelliae by LAMP-LFD[J]. Journal of Agricultural Biotechnology, 24(5): 770-780.) [2] 贺春萍, 李锐, 吴伟怀, 等. 2013. 12种杀菌剂对橡胶树褐根病菌的毒力测定[J]. 热带作物学报, 34(10): 1987-1990. (He C P, Li R, Wu W H, et al.2013. Evaluation of 12 fungicides for suppression of Phellinus noxius[J]. Chinese Journal of Tropical Crops, 34(10): 1987-1990.) [3] 梁艳琼, 吴伟怀, 李锐, 等. 2016. 橡胶树褐根病菌的分子检测[J]. 热带作物学报, 37(03): 576-581. (Liang Y Q, Wu W H, Li R, et al.2016. Molecular detection of Phellinus noxius in rubber tree[J]. Chinese Journal of Tropical Crops, 37(3): 576-581.) [4] 林石明, 廖富荣, 陈红运, 等. 2012. 台湾褐根病发生情况及研究进展[J]. 植物检疫, 26(6): 54-60. (Lin S M, Liao F R, Chen H Y, et al.2012. Research advances of brown root rot (Phellinus noxius) in Taiwan, China[J]. Plant Quarantine, 26(06): 54-60.) [5] 陆辰晨. 2015. 基于环介导等温扩增技术快速诊断大豆根部主要病害的研究[D]. 博士学位论文, 南京农业大学, 导师: 郑小波, pp. 1-107. (Lu C C.2015. Rapid diagnosis of soybean root diseases based on Loop-mediated isotheral amplication (LAMP)[D]. Thesis for Ph.D., NanJing Agricultural University, Supervisor: Zheng X B, pp. 1-107.) [6] 庞耀珊, 谢芝勋, 谢丽基, 等. 2013. 对虾白斑综合征病毒可视化环介导等温扩增(LAMP)检测技术的建立与应用[J]. 农业生物技术学报, 21(8): 1002-1008. (Pang Y S, Xie Z X, Xie L J, et al.2013. Development and appli-cation of visual loop mediated isothermal amplification(LAMP) assay for shrimp (Penacus orientalis) White spotsyndrome virus[J]. Journal of Agricultural Biotechnology, 21(8): 1002-1008.) [7] 秦绪珍. 2004. PCR的污染及控制(基础)[J]. 山东卫生, 5: 53-54. (Qin X Z.2004. Pollution and control of PCR (Basis)[J]. Shandong Health, 5: 53-54.) [8] 沈肖玲, 易建平, 王荣洲, 等. 2019. 甘薯茎腐病菌Taq Man荧光定量PCR检测方法的建立与应用[J]. 农业生物技术学报, 27(03): 551-563. (Shen X L, Yi J P, Wang R Z, et al.2019. Establishment and application of taq man fluorescent quantitative PCR detection method for sweet potato stem rot[J]. Journal of Agricultural Biotechnology, 27(03): 551-563.) [9] 王贤达, 林雄杰, 胡菡青, 等. 2014.柑橘黄龙病可视化LAMP检测技术的建立及应用[J]. 热带作物学报, 35(5): 918-924. (Wang X D, Lin X J, Fu H Q, et al.2014. Establishment and application of visual loop-mediated amplification assay on Citrus Huanglongbing[J]. Chinese Journal of Tropical Crops, 35(5): 918-924.) [10] 吴家林, 沙丹, 马广源, 等. 2015.沙门氏菌LAMP检测方法的建立[J]. 中国病原生物学杂志, 10(7): 611-614. (Wu J L, Sha D, Ma G Y, et al.2015. E stablishment and use of a loop-mediated isothermal amplification technique to detect Salmonella[J]. Journal of Pathogen Biology,10(7): 611-614.) [11] 吴孟玲, 张东柱, 庄铃木. 2009 .树木褐根病PCR检测技术的建立[J]. 中华林业季刊, 42(2): 239-247. (Wu M L, Zhang D Z, Zhuang L M.2009. Establishment of PCR detection technology for brown root disease of trees[J]. China Forestry Quarterly, 42(2): 239-247.) [12] 张劲蔼, 黄华枝, 毕可可, 等. 2016. 褐根病菌分子检测与生物防治研究进展[J]. 广东园林, 38(6): 74-77. (Zhang J A, Huang H Z, Bi K K, et al .2016. Research progress on molecular detection and biological control of Phellinus noxius[J]. Guangdong Landscape Architecture, 38(6): 74-77.) [13] 赵赛, 李建嫄, 周颖, 等. 2015. 番茄溃疡病菌LAMP快速检测方法的建立[J]. 河北农业大学学报, 38(3): 19-23. (Zhao S, Li J B, Zhou Y, et al.2015. Development of a Loop-mediated isothermal amplification assay for detection of Clavibacter michiganensis subsp.michiganensis[J]. Journal of Agricultural University of HeBei, 38(3): 19-23.) [14] 周杰, 黄文胜, 邓婷婷, 等. 2017. 环介导等温扩增法检测6种转基因大豆[J]. 农业生物技术学报, 25(2): 335-344. (Zhou J, Huang W S, DengT T, et al.2017. Detection of six kinds of genetically modified soybean (Glycine max) by LAMP method[J]. Journal of Agricultural Biotechnology, 25(2): 335-344.) [15] Goto M, Honda E, Ogura A, et al.2009. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue[J]. Biotechniques, 46(3): 167-172. [16] Notomi T, Okayama H, Masubuchi H, et al.2000. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 28(12): E63. [17] Parida M, Sannarangaiah S, Dash P K, et al.2008. Loop mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases[J].Reviews in Medical Virology, 18(6): 407-421. [18] Wong Y P, Othman S, Lau Y L, et al.2018. Loop-mediated isothermal amplification (LAMP): A versatile technique for detection of micro-organisms[J]. Journal of Applied Microbiology. 124(3): 626-643. [19] Shiro Fukuta, Shinro Kato, Keiko Yoshida, et al.2003. Detection of Tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction[J]. Journal of Virological Methods, 112(1-2): 35-40 [20] Shan L, Abdul Haseeb H, Zhang J, et al.2018. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of toxigenic Fusarium temperatum in maize stalks and kernels[J]. International Journal of Food Microbiology, 291(16): 72-78. [21] Tomita N, Mori Y, Kanda H, Notomi T.2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products[J]. Nature Protocols, 3(5): 877-882.
