基于重组酶介导扩增-侧流层析试纸条的褐飞虱快速鉴定方法

doi:10.16819/j.1001-7216.2022.210314

摘要/Abstract

摘要：

【目的】针对测报灯下褐飞虱鉴定费时费力这一问题,建立褐飞虱快速定性鉴定技术。【方法】以褐飞虱、伪褐飞虱及拟褐飞虱为材料,筛选褐飞虱特异性引物对,优化基于重组酶介导扩增和侧流层析试纸条检测方法的快速鉴定体系(RAA-LFD),并分析其温度及模板鲁棒性。【结果】本研究建立的RAA-LFD方法操作简单、速度快、不依赖于任何精密仪器、体温可触发扩增反应,且对粗组织液模板具有很好的鲁棒性。盲样检测结果表明,56个样本从样本获取到结果输出的整个过程可在45 min内完成,准确率可达100%。【结论】本研究建立的褐飞虱RAA-LFD鉴定技术可以用于褐飞虱及其近似种的快速区分,适用于基层植保站或田间地头对样本的现场即时检测。

关键词: 褐飞虱, 伪褐飞虱, 拟褐飞虱, RAA-LFD, 现场即时检测

Abstract:

【Objective】To solve the time- and labor-costing problem for accurate identification of Nilarpavata lugens trapped by light, we aim to establish a fast and convenient qualitative identification method.【Method】At first, we selected a couple of primers which are species specific for N. lugens. Then, we established an identification method based on Rapid Aided Amplification-Lateral Flow Dipstick (RAA-LFD) technology. Finally, we analyzed the temperature and template robustness of this method. 【Result】The RAA-LFD method developed in the present study is convenient and fast, and it does not depend on any sophisticated instrument. Furthermore, this method can amplify effectively with crude tissue fluid as template, and it can be triggered by body temperature. The entire process for 56 specimens, including the crude tissue liquid preparation, isothermal amplification, and LFD detection, can be completed in 45 min with an accuracy of 100%. 【Conclusion】This RAA-LFD method is a time-saving and easy-to-apply molecular diagnostic method for N.lugens identification from its two sibling species. It is really suitable for Point of Care Test (POCT) in plant protection station or in field.

Key words: Nilaparvata lugens, Nilaparvata muiri China, Nilaparvata bakeri (Muir), RAA-LFD, Point of Care Test

罗举, 唐健, 王爱英, 杨保军, 刘淑华. 基于重组酶介导扩增-侧流层析试纸条的褐飞虱快速鉴定方法[J]. 中国水稻科学, 2022, 36(1): 96-104.

LUO Ju, TANG Jian, WANG Aiying, YANG Baojun, LIU Shuhua. A Rapid Detection Assay of Nilaparvata lugens Based on Recombinase Aided Amplification-lateral Flow Dipstick Technology[J]. Chinese Journal OF Rice Science, 2022, 36(1): 96-104.

图/表 8

表1 引物和探针

Table 1 Primers and probes.

引物名称 Primer name	引物及探针序列（5＇-3＇） Primers and probe sequences (5＇-3＇)	片段长度 Amplicon length/bp
ITS1-296-F28	TACAACTAGGTAAGTAAAGGTGCTACAG	247
ITS1-515-R28	TCCTGTGTGCTTGCTGTTAAGTCACAAG	247
COI-387-F28	CCTTTCTAGAGTAACATCCCATTCCGG G	174
COI-533-R28	AATACAGATCAACAGAATAGAGGTAATT	174
COI-387-F28	CCTTTCTAGAGTAACATCCCATTCCGG G	218
COI-577-R28	CAGCTAGAACAGGAAGGGATAGAAGGAG	218
Cytb-133-F29	CTATCAATACACTATACACCAAATATTAG	223
Cytb-326-R30-Bio	Biotin-CTATAAGAATCAACATAATTAAGGTCCCTG
Cytb-nfo-probe	FAM-TTATATTTATTCATACAGGGCGAGGAATTT [THF]CTATGGCTCTTTCCTT-C3-Spacer

表2 样本收集地点

Table 2 Regions of specimen collection in China.

地点 Location	坐标Coordinates		收集年份Collection year	样本数量Number of specimen
地点 Location	东经 Longitude (E)/°	北纬 Latitude (N)/°	收集年份Collection year	褐飞虱 N. lugens	伪褐飞虱 N. muiri	拟褐飞虱 N. bakeri	白背飞虱/灰飞虱 S. furcifera / L. striatellus
广西Guangxi	110.80	24.17	2017	2	2	2	–
广西Guangxi	110.80	24.17	2018	3	3	2	1
四川Sichuan	108.53	28.24	2017	2	2	3	–
四川Sichuan	108.53	28.24	2018	2	2	1	1
湖南Hunan	111.73	27.25	2017	2	2	2	–
湖南Hunan	111.73	27.25	2018	2	4	2	1
浙江Zhejiang	119.95	30.07	2017	4	2	0	–
浙江Zhejiang	119.95	30.07	2018	2	1	2	2

图1 检测用RAA-LFD引物及探针的筛选 A–RAA-LFD扩增引物筛选;B–RAA-LFD检测探针验证。+代表褐飞虱基因组DNA;-代表ddH2O。

Fig. 1. Primer and probe selection for RAA-LFD method. A, Primer selection for RAA-LFD; B, Probe selection for RAA-LFD. +, Genome DNA of N. Lugens; -, ddH2O.

图2 褐飞虱及其两个近似种的Cytb基因多序列比对图 Nl–褐飞虱;Nm–伪褐飞虱;Nb–拟褐飞虱。粗字体序列代表P4引物位置,下划线序列代表探针位置。

Fig. 2. Multiple alignment of Cytb genes from N. lugens and its two sibling species. Nl, N. lugens; Nm, N. muiri; Nb, N. bakeri. Primer sequence were highlighted with bold characters, and the probe sequence was underlined.

图3 基于P4引物对的RAA-LFD方法特异性分析结果 Nl–褐飞虱;Nm–伪褐飞虱;Nb–拟褐飞虱;F–雌成虫;M–雄成虫。

Fig. 3. Specificity analysis of the RAA-LFD method based on P4 primer. Nl, N. lugens; Nm, N. muiri; Nb, N. bakeri; F, Female adult; M, Male adult.

图4 基于P4引物对的RAA-LFD方法灵敏度实验结果

Fig. 4. Sensitivity analysis of the RAA-LFD method based on P4 primer.

图5 褐飞虱RAA-LFD检测方法的适用条件范围试验结果 A–扩增温度分析;B–扩增时长分析。Nl–褐飞虱,Nm–伪褐飞虱,Nb–拟褐飞虱。

Fig. 5. Applicability analysis of the RAA-LFD method based on P4 primer. A, Amplification tempreture analysis; B, Amplification speed analysis. Nl, N. lugens; Nm, N. muiri; Nb, N. bakeri.

图6 不同公司试剂扩增活性的对比结果 Nl–褐飞虱;Nm–伪褐飞虱;Nb–拟褐飞虱。

Fig. 6. Amplification ability analysis between RPA-nfo and RAA-nfo reagents. Nl, N. lugens; Nm, N. muiri; Nb, N. bakeri.

参考文献 36

[1]	Bottrell D G, Schoenly K G. Resurrecting the ghost of green revolutions past: The brown planthopper as a recurring threat to high-yielding rice production in tropical Asia[J]. Journal of Asia-Pacific Entomology, 2012,15:122-140.
[2]	Hu G, Lu F, Zhai B P, Lu M H, Liu WC, Zhu F, Wu XW, Chen G H, Zhang X X. Outbreaks of the Brown Planthopper Nilaparvata lugens (Stal) in the Yangtze River Delta: Immigration or local reproduction?[J]. Plos One, 2014,9.
[3]	Xue J, Zhou X, Zhang C X, Yu L L, Fan H W, Wang Z, Xu H J, Xi Y, Zhu Z R, Zhou W W, Pan P L, Li B L, Colbourne J K, Noda H, Suetsugu Y, Kobayashi T, Zheng Y, Liu S, Zhang R, Liu Y, Luo Y D, Fang D M, Chen Y, Zhan D L, Lv X D, Cai Y, Wang Z B, Huang H J, Cheng R L, Zhang X C, Lou Y H, Yu B, Zhuo J C, Ye Y X, Zhang W Q, Shen Z C, Yang H M, Wang J, Wang J, Bao Y Y, Cheng J A. Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation[J]. Genome Biology, 2014,15(12):521.
[4]	Wen Y C, Liu Z W, Bao H B, Han Z J. Imidacloprid resistance and its mechanisms in field populations of brown planthopper, Nilaparvata lugens Stål in China[J]. Pesticide Biochemistry And Physiology, 2009,94(1):36-42.
[5]	崔亚丽, 何佳春, 罗举, 赖凤香, 傅强. 褐飞虱近似种伪褐飞虱和拟褐飞虱的寄主植物[J]. 中国水稻科学, 2013,27(1):105-110.
	Cui Y L, He J C, Luo J, Lai F X, Fu Q. Host plants of Nilaparvala muiri China and N. bakeri (Muir), two sibling species of N. lugens (Stål)[J]. Chinese Journal of Rice Science, 2013,27(1):105-110. (in Chinese with English abstract)
[6]	罗举, 傅强, 陆志坚, 吴彩谦, 李一波, 段德康, 刘玉坤, 张志涛. 测报灯下褐飞虱及其两种近似种的数量动态[J]. 中国水稻科学, 2010,24(3):315-319.
	Luo J, Fu Q, Lu Z J, Wu C Q, Li Y B, Duan D K, Liu Y K, Zhang Z T. Population dynamics of Nilaparvata lugens and its two sibling species under black light trap[J]. Chinese Journal of Rice Science, 2010,24(3):315-319. (in Chinese with English abstract)
[7]	蒋天梅, 虞根聪, 赖朝晖, 蒋晔. 象山县测报灯下褐飞虱及其近似种的数量动态[J]. 中国植保导刊, 2018,38(7):43-45.
	Jiang T M, Yu G C, Lai C H, Jiang Y. Population dynamics of Nilaparvata lugens and its two sibling species under light trap in Xiangshan, Zhejiang Province[J]. China Plant Protection, 2018,38(7):43-45. (in Chinese)
[8]	丁锦华. 褐飞虱属分类及其种类的研究[J]. 南京农业大学学报, 1981,1:78-85.
	Ding J H. A review of studies on classification and species of the Genus Nilaparvata Distant (Homoptera:Delphacidae)[J]. Journal of Nanjing Agricultural College, 1981,1:78-85. (in Chinese with English abstract)
[9]	芮金富, 周建平, 石磊. 褐飞虱与拟褐飞虱和伪褐飞虱的识别[J]. 现代农业科技, 2011,5:191.
	Rui J F, Zhou J P, Shi L. Morphological identification of Nilaparvata lugens from its two sibling species, Nilaparvata muiri and Nilaparvata bakeri[J]. Modern Agriculture Technology, 2011,5:191. (in Chinese)
[10]	崔亚丽. 褐飞虱及其两近似种的生物学及分子标记的研究[D]. 北京:中国农业科学院, 2012: 26-39.
	Cui Y L. Studies on the biological characteristics and molecular markers for Nilaparvata lugens (Stål) and its two sibling species N. muiri China and N. bakeri (Muir) [D]. Beijing: Chinese Academy of Agricultural Sciences, 2012: 26-39. (in Chinese with English abstract)
[11]	Liu S H, Luo J, Liu R, Zhang C G, Duan D K, Chen H M, Bei W Y, Tang J. Identification of Nilaparvata lugens and its two sibling species (N. bakeri and N. muiri) by direct multiplex PCR[J]. Journal of Economic Entomology, 2018,111(6):2869-2875.
[12]	Piepenburg O, Williams C H, Stemple D L, Armes N A. DNA detection using recombination proteins[J]. Plos Biology, 2006,4(7):1115-1121.
[13]	吕蓓, 程海荣, 严庆丰, 黄震巨, 沈桂芳, 张志芳, 李轶女, 邓子新, 林敏, 程奇. 用重组酶介导扩增技术快速扩增核酸[J]. 中国科学: 生命科学, 2010,40(17):983-988.
	Lv B, Cheng H R, Yan Q F, Huang Z J, Shen G F, Zhang Z F, Li Y N, Deng Z X, Lin M, Cheng Q. Rapid neclic acid amplification with recombinase aided amplification technology[J]. Scientia Sinica Vitae, 2010,40(17):983-988. (in Chinese)
[14]	孙魁, 邢微微, 徐东刚, 宋伦. 基于重组酶聚合酶扩增结合侧流层析技术的B、E型腺病毒快速可视化检测方法的研究与评价[J]. 军事医学, 2017,41(7):547-551.
	Sun K, Xing W W, Xu D G, Song L. Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of adenovirus[J]. Military Medical Sciences, 2017,41(7):547-551. (in Chinese with English abstract)
[15]	赵松, 刘燕红, 李婷, 李伟, 张键锋, 郭利川, 应清界, 羊海涛, 杨坤. 结合重组酶介导的核酸等温扩增和荧光探针快速检测日本血吸虫基因片段[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(1):23-27.
	Zhao S, Liu Y H, Li T, Li W, Zhang J F, Guo L C, Ying Q J, Yang H T, Yang K. Rapid detection of Schistosoma japonicum specific gene fragment by recombinase aided isothermal amplification combined with fluorescent probe[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019,37(1):23-27. (in Chinese with English abstract)
[16]	李樊, 郭晓芳, 殷启凯, 申辛欣, 付士红, 许松涛, 雷雯雯, 周红宁, 马学军, 王环宇. 塔希纳病毒的重组酶介导扩增快速检测方法[J]. 疾病监测, 2020,35(3):192-196.
	Li F, Guo X F, Yin Q K, Shen X X, Fu S H, Xu S T, Lei W W, Zhou H N, Ma X J, Wang H Y. Establishment of a reverse transcription recombinase-mediated amplification assay for rapid detection of Tahyna virus[J]. Disease Surveillance, 2020,35(3):192-196. (in Chinese with English abstract)
[17]	Lv L, Peng X X, Jing S L, Liu B F, Zhu L L, He G C. Intraspecific and interspecific variations in the mitochondrial genomes of Nilaparvata (Hemiptera: Delphacidae)[J]. Journal of Economic Entomology, 2015,108(4):2021-2029.
[18]	Li J, Macdonald J, von Stetten F. Review: A comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst, 2019,144(1):31-67.
[19]	Wee E J H, Lau H Y, Botella J R, Trau M. Re-purposing bridging flocculation for on-site, rapid, qualitative DNA detection in resource-poor settings[J]. Chemical Communications, 2015,51(27):5828-5831.
[20]	樊晓旭, 宋翥远, 赵永刚, 赵明, 董雅琴, 张永强, 李园丽, 迟田英, 刘春菊, 戈胜强, 张志诚, 吴晓东, 王树双, 王志亮. 塞尼卡谷病毒重组酶聚合酶扩增-侧流层析试纸条检测方法的建立[J]. 中国预防兽医学报, 2018,40(5):406-410.
	Fan X X, Song Z Y, Zhao Y G, Zhao M, Dong Y Q, Zhang Y Q. Li Y L, Chi T Y, Liu C J, Ge S Q, Zhang Z C, Wu X D, Wang S S, Wang Z L. Establishment of recombinase polymerase amplification-lateral flow dipstick for the detection of Seneca valley virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2018,40(5):406-410. (in Chinese with English abstract)
[21]	葛志毅, 周建华, 尚佑军, 李学瑞, 刘永生, 赵鹭, 孙晶晶, 曹小安. 一种侧流试纸结合FAM重组酶聚合酶扩增(RPA)诊断布鲁氏菌感染的方法[J]. 中国人兽共患病学报, 2019,35(10):905-908.
	Ge Z Y, Zhou J H, Shang Y J, Li X R, Liu Y S, Zhao L, Sun J J, Cao X A. Lateral flow test strips combined with FAM recombinase polymerase amplification ( RPA) for the diagnosis of Brucella infection[J]. Chinese Journal of Zoonoses, 2019,35(10):905-908. (in Chinese with English abstract)
[22]	兰全学, 陈佳平, 杨慧, 杜文旗, 金玉娟, 甘莉萍, 刘渠, 刘小青. 食品中空肠弯曲菌荧光重组酶聚合酶扩增检测方法的建立[J]. 现代食品科技, 2020,36(5) : 1-6.
	Lan Q X, Chen J P, Yang H, Du W Q, Jin Y J, Gan L P, Liu Q, Liu X Q. Development of a detection method for food-borne Campylobacter jejuni based on fluorescence probe-based recombinase polymerase amplification[J]. Modern Food Science and Technology, 2020,36(5):1-6. (in Chinese with English abstract)
[23]	柳海宾, 张海鑫, 宋腾飞, 刘聪, 聂付磊. 环介导等温扩增结合免疫层析试纸条快速检测羊肉及其制品中的鸭源成分 [J]. 食品工业科技, 2020, 41(16): 238-241+251.
	Liu H B, Zhang H X, Song T F, Liu C, Nie F L. Rapid detection of duck derived components in mutton and mutton products by loop mediated isothermal amplification combined with immune chromatographic test strip [J]. Science and Technology of Food Industry, 2020, 41(16): 238-241+251. (in Chinese with English abstract)
[24]	袁伟, 廖凌, 童淑梅, 康晓平. 国境口岸病原体检测新方向-重组酶聚合酶扩增技术[J]. 中国国境卫生检疫杂志, 2019,42(4):295-300.
	Yuan W, Liao L, Tong S M, Kang X P. New direction for pathogens detection at national frontier ports: Recombinase polymerase amplification technology[J]. Chinese Frontier Health Quarantine, 2019,42(4):295-300. (in Chinese with English abstract)
[25]	冯黎霞, 魏霜, 余辛, 张永江, 武目涛, 胡学难. 重组酶聚合酶扩增技术(RPA)快速检测玉米褪绿斑驳病毒[J]. 植物保护学报, 2020,47(1):217-218.
	Feng L X, Wei S, Yu X, Zhang Y J, Wu M T, Hu X N. Detection of maize chlorotic mottle virus by recombinase polymerase amplification[J]. Journal of Plant Protection, 2020,47(1):217-218. (in Chinese)
[26]	谢实龙, 汪小福, 丁晨露, 祝旋, 汤婷, 马同富, 蔡健, 徐俊锋. 转基因大豆MON89788实时荧光重组酶聚合酶扩增检测方法的建立[J]. 农业生物技术学报, 2019,27(7):1301-1310.
	Xie S L, Wang X F, Ding C L, Zhu X, Tang T, Ma T F, Cai J, Xu J F. Establishment of a real-time fluorescence recombinase polymerase amplification for detection of transgenic soybean (Glycine max) MON89788[J]. Journal of Agricultural Biotechnology, 2019,27(7):1301-1310. (in Chinese with English abstract)
[27]	王燕, 王春伟, 王琳, 荆琦, 高海馨, 余廷濠, 王美琴, 张作刚, 王建明. 樱桃灰霉病菌LFD-RPA快速检测方法的建立[J]. 植物病理学报, 2020,50(2):238-245.
	Wang Y, Wang C W, Wang L, Jing Q, Gao H X, Yu T H, Wang M Q, Zhang Z G, Wang J M. Development of LFD-RPA assay for rapid detection of Botrytis cinerea in cherry[J]. Acta Phytopathologica Sinica, 2020,50(2):238-245. (in Chinese with English abstract)
[28]	许笑, 王艳华, Khan M Z, 王恒, 蔡建平. 鸡源产气荚膜梭菌重组酶聚合酶扩增检测方法的建立[J]. 中国兽医科学, 2019,49(12):1475-1483.
	Xu X, Wang Y H, Khan M Z, Wang H, Cai J P. Recombinase polymerase amplification: A detection method for the detection of Clostridium perfringens from chicken[J]. Chinese Veterinary Science, 2019,49(12):1475-1483. (in Chinese with English abstract)
[29]	彭遥, 阚飙, 夏连续, 李伟, 张雯, 秦爱平, 卢金星. 侧流层析-重组酶聚合酶扩增技术快速检测土拉弗朗西斯菌[J]. 疾病监测, 2019,34(5):455-459.
	Peng Y, Kan B, Xia LX, Li W, Zhang W, Qin AP, Lu JX. Establishment of a recombinase polymerase amplification combining with lateral flow strip assay for rapid visual detection of Francisella tularensis[J]. Disease Surveillance, 2019,34(5):455-459. (in Chinese with English abstract)
[30]	Gootenberg J S, Abudayyeh O O, Kellner M J, Joung J, Collins J J, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science, 2018,360:439-444.
[31]	Chen J S, Ma E B, Harrington L B, Da Costa M, Tian X R, Palefsky J M, Doudna J A. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity [J]. Science, 2018, 360(6387): eaar6245.
[32]	Wang B, Wang R, Wang D Q, Wu J, Li J X, Wang J, Liu H H, Wang Y M. Cas12aVDet: A CRISPR/Cas12a-based platform for rapid and visual nucleic acid detection[J]. Analytical Chemistry, 2019,91(19):12156-12161.
[33]	赵巧雅, 马秀丽, 刘存霞, 史玉颖, 胡峰, 王贵升, 兰邹然, 黄兵. 兔源肺炎克雷伯氏菌重组酶聚合酶扩增检测方法的建立[J]. 中国动物检疫, 2019,36(7):82-85.
	Zhao Q Y, Ma X L, Liu C X, Shi Y Y, Hu F, Wang G S, Lan Z R, Huang B. Establishment of a RPA assay for detecting klebsiella pneumoniae of rabbit origin[J]. China Animal Health Inspection, 2019,36(7):82-85. (in Chinese with English abstract)
[34]	王秋平, 黄恩奇, 杨银梅, 钟志敏, 陈斌. 应用直接重组酶聚合酶扩增技术快速检测烟曲霉菌[J]. 中华医院感染学杂志, 2018,28(24):3734-3737.
	Wang Q P, Huang E Q, Yang Y M, Zhong Z M, Chen B. Rapid detection of Aspergillus fumigatus by direct recombinant polymerase amplification[J]. Chinese Journal of Nosocomiology, 2018,28(24):3734-3737. (in Chinese with English abstract)
[35]	Crannell Z, Castellanos-Gonzalez A, Nair G, Mejia R, White A C, Richards-Kortum R. Multiplexed recombinase polymerase amplification assay to detect intestinal protozoa[J]. Analytical Chemistry, 2016,88(3):1610-1616.
[36]	Lau H Y, Wang Y, Wee E J, Botella J R, Trau M. Field demonstration of a multiplexed point-of-care diagnostic platform for plant pathogens[J]. Analytical Chemistry, 2016,88(16):8074-8081.