玉米根腐病菌麦根腐平脐蠕孢的LAMP检测方法

doi:10.7668/hbnxb.20193347

摘要/Abstract

摘要：

麦根腐平脐蠕孢是玉米根腐病的重要病原菌之一,旨为建立一种基于环介导等温扩增技术(LAMP)的麦根腐平脐蠕孢快速检测方法。首先,根据麦根腐平脐蠕孢参与黑色素生物合成途径的Brn1基因部分序列设计了一套LAMP检测引物;然后,针对该引物组筛选了其最佳反应温度,检测了LAMP反应的特异性和灵敏度,并以人工接种麦根腐平脐蠕孢的玉米根腐病病株为样本评价了LAMP检测方法的效果。结果表明,本研究设计的引物组在61~68 ℃条件下均能实现对靶标基因的扩增,其中以66 ℃为最佳反应温度;在特异性检测中,引物组能从10种分离自玉米根腐病样本的主要病原真菌DNA中特异性地检测出麦根腐平脐蠕孢;在灵敏度检测中,对携带靶基因Brn1的质粒DNA最低检测限是10 copies/μL,在此检测限浓度下,25 min左右即可实现扩增;在对玉米根腐病样本检测中,在1 pg/μL的玉米根组织DNA中即可检测出麦根腐平脐蠕孢。建立的麦根腐平脐蠕孢LAMP检测方法具有较强的特异性和较高的灵敏度。

关键词: 玉米根腐病, 麦根腐平脐蠕孢, Brn1基因, 环介导等温扩增, 检测方法

Abstract:

Bipolaris sorokiniana is one of the important pathogens of maize root rot.The purpose of the present work was to establish a rapid detection method for B.sorokiniana based on the loop-mediated isothermal amplification(LAMP).Firstly,a set of primers for LAMP assay was designed according to the partial sequence of Brn1 involved in the melanin biosynthetic pathway.Then,the optimal reaction temperature was screened for this primer set,the specificity and sensitivity of LAMP reaction were detected,and the LAMP detection was evaluated by using the maize root rot samples artificially inoculated with B.sorokiniana.The results showed that the primer set designed could amplify the target gene Brn1 at 61—68 ℃,with 66 ℃ was the optimal temperature.In the specific detection,the primer set could specifically detect B.sorokiniana from the genomic DNA of 10 main pathogenic fungi isolated from maize root rot plants.In the sensitivity detection,the minimum detection limit for plasmid DNA carrying Brn1 was 10 copies/μL,and amplification could be achieved in about 25 min at the minimum concentration.And,for the detection of maize root rot samples,B.sorokiniana can be detected in 1 pg/μL of maize root tissue DNA.These results indicated that the LAMP detection method for B.sorokiniana established in this study has robust specificity and high sensitivity.

Key words: Maize root rot, Bipolaris sorokiniana, Brn1 gene, LAMP, Detection method

刘树森, 郭宁, 孙华, 马红霞, 张海剑, 石洁. 玉米根腐病菌麦根腐平脐蠕孢的LAMP检测方法[J]. 华北农学报, 2023, 38(1): 162-167. doi: 10.7668/hbnxb.20193347.

LIU Shusen, GUO Ning, SUN Hua, MA Hongxia, ZHANG Haijian, SHI Jie. Loop-Mediated Isothermal Amplification Assay for Detection of Bipolaris sorokiniana Causing Maize Root Rot[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 162-167. doi: 10.7668/hbnxb.20193347.

http://www.hbnxb.net/CN/Y2023/V38/I1/162

图/表 6

表1 本研究所用菌株

Tab.1 Isolates used in this study

真菌菌株 Fungi isolate	寄主 Host	采集地 Collection site
麦根腐平脐蠕孢Bipolaris sorokiniana	玉米根部	山东省淄博市高青县
玉米生平脐蠕孢Bipolaris zeicola	玉米根部	承德市宽城满族自治县
嘴突凸脐蠕孢Exserohilum rostratum	玉米根部	河南省新乡市红旗区
链隔孢Alternaria alternata	玉米根部	河南省驻马店市泌阳县
弯孢菌Curvularia sp.	玉米根部	山东省菏泽市定陶区
小柄凸脐蠕孢Exserohilum pedicellata	玉米根部	河北省唐山市丰南区
拟轮枝镰孢Fusarium verticillioides	玉米根部	河南省安阳市安阳县
层出镰孢Fusarium proliferatum	玉米根部	河南省孟州市
禾谷镰孢Fusarium graminearum	玉米根部	山东省济南市长青区
尖孢镰孢Fusarium oxysporum	玉米根部	山东省临沂市

表2 本研究所用引物序列

Tab.2 Primer sequences used in this study

引物名称 Primer name	序列(5'—3') Sequence (5'—3')	作用 Function	文献 Reference
Brn01	GCCAACATCGAGCAAACATGG	Brn1扩增	[17]
Brn02	GCAAGCAGCACCGTCAATACCAAT
BSF3	GAGGAGTTCGACCGTGTC
BSB3	GCACCTGGTGAAAGTCTCA	LAMP	本研究
BSFIP	CATCTCCATGCGCTTGTACGC
	CACCATCAACACTCGTGG
BSBIP	ATCCTCATGGGATCCATCACCG
	TTTGAGCCAGAGTAGACAGC
BSLF	TTGGCAACGAAGAACTGA
BSLB	GTCAGGCAAAGGGTGT

图1 不同温度条件下的扩增曲线

Fig.1 Amplification curves at different temperatures

图2 LAMP反应特异性扩增曲线和可视化检测 A.LAMP反应特异性扩增曲线;B.LAMP反应特异性可视化检测:a.麦根腐平脐蠕孢基因组DNA,b.携带Brn1的质粒DNA,c~l.嘴突凸脐蠕孢、链隔孢、弯孢菌、小柄凸脐蠕孢、拟轮枝镰孢、层出镰孢、禾谷镰孢、尖孢镰孢、玉米生平脐蠕孢、无菌水。

Fig.2 Amplification curves and visual inspection of the LAMP specificity A.Amplification curves of the LAMP specificity;B.Visual inspection of the LAMP specificity:a.Bipolaris sorokiniana genomic DNA,b.Plasmid DNA containing Brn1 gene,c—l.Exserohilum rostratum,Alternaria alternata,Curvularia sp.,Exserohilum pedicellata,Fusarium verticillioides,Fusarium proliferatum, Fusarium graminearum, Fusarium oxysporum, Bipolaris zeicola and sterilized distilled water.

图3 LAMP反应灵敏度扩增曲线和可视化检测 A.LAMP反应灵敏度扩增曲线;B.LAMP反应灵敏度可视化检测:a~g.10⁵,10⁴,10³,10²,10¹,10⁰,10^-1 copies/μL模板DNA;h.阴性对照。

Fig.3 Amplification curves and visual inspection of the LAMP sensitivity A.Amplification curves of the LAMP sensitivity;B.Visual inspection of the LAMP sensitivity:a—g.10⁵,10⁴,10³,10²,10¹,10⁰,10^-1 copies/μL template DNA;h.Negative control.

图4 LAMP可视化检测玉米根腐病样本中的麦根腐平脐蠕孢 a~g.100 ng/μL,10 ng/μL,1 ng/μL,100 pg/μL,10 pg/μL,1 pg/μL,100 fg/μL模板DNA;h.阴性对照。

Fig.4 Visual inspection of B.sorokiniana in diseased maize root by LAMP a—g.100 ng/μL,10 ng/μL,1 ng/μL,100 pg/μL,10 pg/μL,1 pg/μL,100 fg/μL of DNA;h.Negative control.

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