植物青枯病菌环介导等温扩增快速检测技术研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0389

摘要/Abstract

摘要：

为实现植物青枯病的早期诊断,需要建立一种适于田间快速便捷检测青枯病菌的方法。以细胞色素C基因为靶标设计一套特异性引物,建立了植物青枯病的LAMP检测方法。此方法最低检测极限为1 pg,可在1 h 内完成,不依赖昂贵复杂的仪器,结果可经肉眼观察。利用此方法,在人工接种发病的茄子、番茄、花生、芝麻和凹头苋茎部浸出液和马铃薯病薯块茎组织液中均检测出青枯病菌的存在,尤其适用于田间疑似罹病的芝麻、花生、番茄、马铃薯和甘薯等植株的检测,且LAMP法的检出率远高于PCR法。应用LAMP技术检测青枯病菌快速高效、特异性强、灵敏度高,操作简单,适于在基层推广运用。

关键词: 青枯雷尔氏菌, 环介导等温扩增, 快速检测

Abstract:

The purpose of the study is to establish a rapid detection method for Ralstonia solanacearum,which is beneficial to the early diagnosis of plant bacterial wilt in the field. The LAMP（loop-mediated isothermal amplification）method for amplification of cytochrome C gene was established by optimization of reaction conditions. The minimum detection limit with the LAMP method reached 1 pg. The detection could be completed within 1 h without using complicated instruments and the results were visible by human eye. Using this method,R. solanacearum was detected in the stem extracts of manually inoculated eggplant,tomato,peanut,sesame and Amaranthus lividus and potato tuber tissue fluid of potato,especially,it was appropriate for the detection of sesame,peanut,tomato,potato and sweet potato plants suspected of being in disease in the field,and the detection rate by LAMP was much higher than that by PCR method. The LAMP method is a fast,specific,sensitive,and easy-to-use for the field detection of R. solanacearum bacterial wilt,which is suitable for promotion at the grassroots level.

Key words: Ralstonia solanacearum, loop-mediated isothermal amplification, rapid detection

李信申, 黄小梅, 吴淑秀, 黄瑞荣, 魏林根, 华菊玲. 植物青枯病菌环介导等温扩增快速检测技术研究[J]. 生物技术通报, 2021, 37(1): 272-281.

LI Xin-shen, HUANG Xiao-mei, WU Shu-xiu, HUANG Rui-rong, WEI Lin-gen, HUA Ju-ling. Rapid Detection of Plant Bacterial Wilt by Loop-mediated Isothermal Amplification[J]. Biotechnology Bulletin, 2021, 37(1): 272-281.

图/表 8

表1 用于检测引物特异性的青枯病菌和非青枯病菌菌株

菌株编号	菌株名称	寄主	演化型/生理小种
1	青枯病菌菌株Ejxnc01	茄子	Ⅰ/1
2	青枯病菌菌株Cjxnc01	辣椒	Ⅰ/1
3	青枯病菌菌株Tjxjx01	番茄	Ⅰ/1
4	青枯病菌菌株Pjxga01	花生	Ⅰ/1
5	青枯病菌菌株Seppx05	芝麻	Ⅰ/1
6	青枯病菌菌株JSja03	凹头苋	Ⅰ/1
7	青枯病菌菌株Tjxga03	烟草	Ⅰ/1
8	青枯病菌菌株Gjxjj01	姜	Ⅰ/4
9	青枯病菌菌株Pjxnc01	马铃薯	Ⅱ/3
10	青枯病菌菌株Pjxga02	马铃薯	Ⅱ/3
11	青枯病菌菌株Pjxjj03	马铃薯	Ⅱ/3
12	青枯病菌菌株Pjxda04	马铃薯	Ⅱ/3
13	蒲桃雷尔氏菌R001	丁香树	-
14	皮氏伯克霍尔德氏菌Bp001	土壤	-

表2 用于环式扩增细胞色素C信号肽的引物序列

引物名称	引物序列（5'-3'）	长度/bp
F3	AGCGGTGCCAATCCGTA	17
B3	TGCCATGGTCAGGTACTGAT	20
FIP	AGCAATCCGAAGGTGCCGAATGTCGCGTA- CAACCAGGA	38
BIP	TCGGTATCCCGACAACACCATGTGGGCGT- CGATCGCATA	39

图1 LAMP反应体系的优化（A）M：Marker DL2000;1：0 mmol/L;2：2 mmol/L;3：4 mmol/L;4：6 mmol/L;5：8 mmol/L;6：10 mmol/L;7：12 mmol/L;CK：阴性对照;（B）M：Marker DL2000;1-6.内引物浓度/外引物浓度分别为2∶1,4∶1,6∶1,8∶1,10∶1,12∶1;CK：阴性对照;（C）M：Marker DL2000;1：59℃;2：60℃;3：61℃;4：62℃;5：63℃;6：64℃;7：65℃;CK：阴性对照

图2 LAMP反应的特异性 1-12：青枯病菌菌株;13-14：非青枯病菌菌株;15：阴性对照

图3 LAMP反应的灵敏度 A：LAMP电泳结果和可视化结果;B：PCR电泳结果。1-7：模板DNA浓度分别为100 ng/μL、10 ng/μL、1 ng/μL、100 pg/μL、10 pg/μL、1 pg/μL、100 fg/μL;CK：阴性对照2.4 人工接种植物青枯病样品LAMP 检测

图4 茄子、番茄、花生、芝麻和凹头苋罹病组织液中青枯菌的LAMP检测 A：茄子;B：番茄;C：花生;D：芝麻;E：凹头苋

图5 马铃薯罹病样品组织液中青枯菌的LAMP检测

表3 疑似罹病植株样本LAMP和PCR检测结果

来源植物	取样部位	样本数量	LAMP检测		PCR检测
来源植物	取样部位	样本数量	检出总数	检出率	检出总数	检出率
芝麻	茎杆	25	25	100%	23	92.00%
花生	茎杆	24	23	95.83%	21	87.50%
番茄	叶片	23	23	100%	21	91.30%
马铃薯	块茎	24	23	95.83%	22	91.67%
甘薯	块茎	22	21	95.45%	20	90.91%

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