基于LAMP的果蔬斯高维尔炭疽菌可视化检测方法

doi:10.3969/j.issn.1004-1524.2022.01.12

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (1): 98-103.DOI: 10.3969/j.issn.1004-1524.2022.01.12

基于LAMP的果蔬斯高维尔炭疽菌可视化检测方法

罗跃^a^,^b(), 吴小毛^a^,^b^,^*(), 刘阿丽^a^,^b, 姚小龙^a^,^b, 李荣玉^a^,^b, 耿广东^b

贵州大学a.作物保护研究所
b.农学院,贵州贵阳 550025

收稿日期:2020-12-24 出版日期:2022-01-25 发布日期:2022-02-05
通讯作者: 吴小毛
作者简介:* 吴小毛,E-mail: wuxm827@126.com
罗跃(1996—),男,贵州水城人,硕士研究生,研究方向为农药科学应用与风险评估、农药残留与环境行为、农产品质量与安全评价。E-mail: 1987957869@qq.com
基金资助:
贵州省科技支撑计划(黔科合支撑〔2019〕2272);贵州省科技支撑计划(黔科合支撑〔2020〕1Y134);贵州省科技成果应用及产业化计划(黔科合成果〔2021〕059);贵州省科技平台及人才团队建设计划(黔科合平台人才〔2018〕5219);贵州省高层次创新型人才培养(百层次创新型人才)(黔科合平台人才〔2022〕);贵州大学培育项目(贵大培育〔2019〕09)

Visual detection method of Colletotrichum scovilleiin fruit and vegetable based on LAMP

LUO Yue^a^,^b(), WU Xiaomao^a^,^b^,^*(), LIU Ali^a^,^b, YAO Xiaolong^a^,^b, LI Rongyu^a^,^b, GENG Guangdong^b

a. Institute of Crop Protection
b.College of Agriculture, Guizhou University, Guiyang 550025, China

Received:2020-12-24 Online:2022-01-25 Published:2022-02-05
Contact: WU Xiaomao

摘要/Abstract

摘要：

为果蔬斯高维尔炭疽菌检测提供高效便捷、特异性好、可视化的检测方法,以果蔬斯高维尔炭疽菌内部转录间隔区ITS序列为靶基因,设计4条LAMP引物和1条环引物,通过对引物比例、反应时间、反应体系等条件的选择,建立对果蔬斯高维尔炭疽菌具有特异性扩增的LAMP检测方法。特异性试验结果显示:所建立的LAMP反应体系在最佳反应温度65 ℃条件下扩增60 min,以果蔬斯高维尔炭疽菌DNA为模板的反应体系在指示剂钙黄绿素和羟基萘酚蓝的作用下,分别呈现绿色和蓝色,反应结果为阳性,扩增产物经2%琼脂糖凝胶电泳检测呈梯形条带;而以其他8株对照菌株DNA为模板的反应体系和空白对照分别呈现橘黄色和紫色,均为阴性反应,无扩增条带出现,表明该LAMP检测方法对果蔬斯高维尔炭疽菌特异性强。

关键词: 环介导等温扩增技术, 果蔬, 斯高维尔炭疽菌, 可视化检测

Abstract:

To provide an efficient, convenient, specific and visual detection method for the detection of Colletotrichum scovillei of fruit and vegetable, taking the ITS sequence of the internal transcription spacer of C.scovillei of fruit and vegetable as the target gene, 4 LAMP primers and 1 loop primer were designed. By choice the primer ratio, reaction time, reaction system and other conditions, a LAMP detection method with specific amplification for C. scovillei was established. The results of the specificity test show that the established LAMP reaction system was amplified for 60 min at the optimal reaction temperature of 65 ℃. The reaction system using C. scovillei DNA as a template was used as the indicator calcein and hydroxynaphthol. Under the action of blue, green and blue appear, respectively. The reaction result was positive, and the amplified product was detected as a ladder band by 2% agarose gel electrophoresis. The reaction system and blank control using the DNA of other 8 control strains as templates,they were orange and purple respectively, and both were negative reactions, and no amplified bands appeared, indicating that the LAMP detection method is highly specific to C.scovillei of fruit and vegetable.

Key words: LAMP, fruit and vegetable, Colletotrichum scovillei, visual detection

中图分类号:

S432.9

罗跃, 吴小毛, 刘阿丽, 姚小龙, 李荣玉, 耿广东. 基于LAMP的果蔬斯高维尔炭疽菌可视化检测方法[J]. 浙江农业学报, 2022, 34(1): 98-103.

LUO Yue, WU Xiaomao, LIU Ali, YAO Xiaolong, LI Rongyu, GENG Guangdong. Visual detection method of Colletotrichum scovilleiin fruit and vegetable based on LAMP[J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 98-103.

图/表 5

表1 引物序列

Table 1 Primer sequence

引物名称 Primer name	序列 Sequence(5'→ 3')
F3	AGCATTCTGGCGAGCATG
B3	TGATCCGAGGTCAACCTGTA
FIP	CCTTTAAGGGCCCACGTGTGCAGCGTCATTT-CAACCCTCAA
BIP	CGGAGCCTCCTTTGCGTAGTAAGGGTTTTAC-GGCAAGAGTCC
LB	CTAACGTCTCGCACTGGGAT

图1 LAMP体系不同反应温度和时间的扩增结果 M:DL2000 marker。a:LAMP体系不同反应温度的扩增结果;1,阴性对照;2,60 ℃;3,62 ℃;4,64 ℃;5,65 ℃;6,66 ℃。b:LAMP体系不同反应时间的扩增结果;1,阴性对照;2,30 min;3,40 min;4,50 min;5,60 min;6,70 min。

Fig.1 Amplification results of LAMP system at different reaction temperature and time M:DL2 000 marker. a: Amplification results of LAMP system at different reaction temperatures; 1, negative control; 2, 60 ℃;3, 62 ℃; 4, 64 ℃; 5, 65 ℃; 6, 66 ℃.b: Amplification result of LAMP system with different reaction times; 1, negative control; 2,30 min; 3,40 min; 4,50 min; 5,60 min; 6, 70 min.

图2 LAMP反应体系检测斯高维尔炭疽菌 a:LAMP产物琼脂糖凝胶电泳检测结果;b:LAMP产物羟基萘酚蓝显色结果;c:LAMP产物钙黄绿素荧光显色结果。1,阴性对照(-);2,斯高维尔炭疽菌(+);3,斯高维尔炭疽菌(+);4,阴性对照(-)。

Fig.2 LAMP reaction system for detecting Colletotrichum scovillei a: LAMP product agarose gel electrophoresis detection results; b: LAMP product hydroxynaphthol blue color results; c: LAMP product calcein fluorescence color results. 1, negative control (-); 2, Colletotrichum scovillei (+);3, Colletotrichum scovillei (+); 4, negative control (-).

图3 钙黄绿素指示下LAMP方法的特异性 M:DL2000 DNA marker。a: LAMP产物钙黄绿素荧光显色结果; b: LAMP产物琼脂糖凝胶电泳检测结果。1,阴性对照;2~4,斯高维尔炭疽菌;5,胶孢炭疽菌;6~8,喀斯特炭疽菌;9~10,水稻稻瘟病菌;11~12,刺梨叶斑病菌。

Fig.3 Specificity of LAMP method under the indication of calcei M: DL2000 DNA marker。a: LAMP product calcein fluorescence coloring result; b: LAMP product agarose gel electrophoresis detection result. 1, Negative control; 2-4, Colletotrichum scovillei; 5, Colletotrichum gloeosporioides; 6-8, Colletotrichum karstii; 9-10, Magnaporthe oryzae; 11-12, Aclinonema sp.

图4 羟基萘酚蓝指示下LAMP方法的特异性 M:DL2000 DNA marker。a: LAMP产物羟基萘酚蓝显色结果;b: LAMP产物琼脂糖凝胶电泳检测结果。1,阴性对照;2~4,斯高维尔炭疽菌;5,胶孢炭疽菌;6~8,喀斯特炭疽菌;9~10,水稻稻瘟病菌;11~12,刺梨叶斑病菌。

Fig.4 Specificity of LAMP method under the indication of hydroxynaphthol blue M: DL2000 DNA marker。 a: LAMP product hydroxynaphthol blue color result; b: LAMP product agarose gel electrophoresis detection result. 1, Negative control; 2-4,Colletotrichum scovillei; 5, Colletotrichum gloeosporioides; 6-8, Colletotrichum karstii; 9-10, Magnaporthe oryzae; 11-12,Aclinonema sp.

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基于LAMP的果蔬斯高维尔炭疽菌可视化检测方法

Visual detection method of Colletotrichum scovilleiin fruit and vegetable based on LAMP

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