三七根腐病菌尖孢镰刀菌实时荧光LAMP检测体系的建立及应用

doi:10.7668/hbnxb.20194307

摘要/Abstract

摘要：

为了实现对三七根腐病菌的快速准确诊断、及时阻断病菌传播、开展针对性防治,建立了一种现场快速检测三七根腐病菌尖孢镰刀菌的实时荧光LAMP技术。首先从GenBank中筛选出尖孢镰刀菌类跨膜蛋白(TP)基因,并针对其保守区域设计了一套LAMP特异性引物,通过对尖孢镰刀菌LAMP检测体系的反应温度、内引物、外引物、环引物、Mg²⁺、dNTPs浓度进行优化,并评价体系的特异性及灵敏性,最后应用建立的LAMP检测方法分析三七病株及土壤样品中尖孢镰刀菌的含量。结果表明,LAMP检测方法最佳反应条件为: 反应温度64 ℃,内引物、外引物以及环引物的最佳浓度分别为1.6,0.2,0.1 μmol/L,Mg²⁺浓度6 mmol/L,dNTPs浓度0.4 mmol/L。利用建立的LAMP检测体系能从7种三七根腐病菌中特异性检测出尖孢镰刀菌,且灵敏度高,检测的最低模板浓度为0.2 pg/μL。此外,将DNA简提法和钙黄绿素指示剂与LAMP检测体系结合,建立了一种三七根腐病菌尖孢镰刀菌的现场快速检测方法,可快速准确检测三七植株及种植土壤中的尖孢镰刀菌,为土壤处理、根腐病的早期诊断和动态监测以及三七种子、种苗流通过程中的带菌分子检测提供技术支持。

关键词: 三七根腐病, 尖孢镰刀菌, 类跨膜蛋白基因, 实时荧光LAMP

Abstract:

The purpose of this study is to develop a loop-mediated isothermal amplification(LAMP)technique for rapid detection of Panax notoginseng root rot pathogen Fusarium oxysporum, timely block the spread of pathogen,and specifically prevent and control root rot.Firstly,F.oxysporum transmembrane protein-like(TP)gene was screened from GenBank,and a set of LAMP specific primers was designed based on its conserved region.In addition,the reaction temperature,and concentrations of inner primer,outer primer,loop primer,Mg²⁺,and dNTPs in the F.oxysporum LAMP detection system were optimized,and then the specificity and sensitivity of this system were evaluated.Finally,the LAMP detection system was used to analyze the F.oxysporum in diseased P.notoginseng plants and soil.The results showed that the optimal reaction temperature for LAMP was 64 ℃,the concentrations of inner primer,outer primer,loop primer,Mg²⁺,and dNTPs were 1.6,0.2,0.1 μmol/L,6 mmol/L,and 0.4 mmol/L,respectively.The LAMP system can specifically detect the F.oxysporum from seven pathogens isolated from P.notoginseng root rot samples with a sensitivity of 0.2 pg/μL.Finally,an on-the-spot detection method for root rot pathogen F.oxysporum in P.notoginseng was developed by combining DNA extraction-free and calcein-green indicator,and this study provided a technical support for convenient detection of F.oxysporum in P.notoginseng planting process,cultivating soil,and seed/seedling trading.

Key words: Panax notoginseng root rot, Fusarium oxysporum, Transmembrane protein-like, Real-time fluorescent LAMP

李有玉, 顾悦, 甘昆发, 崔秀明, 刘迪秋. 三七根腐病菌尖孢镰刀菌实时荧光LAMP检测体系的建立及应用[J]. 华北农学报, 2024, 39(S1): 237-244. doi: 10.7668/hbnxb.20194307.

LI Youyu, GU Yue, GAN Kunfa, CUI Xiuming, LIU Diqiu. Establishment and Application of Real-time Fluorescent LAMP Rapid Detection of Fusarium oxysporum of Panax notoginseng Root Rot[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 237-244. doi: 10.7668/hbnxb.20194307.

http://www.hbnxb.net/CN/Y2024/V39/IS1/237

图/表 6

图1 类跨膜蛋白基因引物特异性检测尖孢镰刀菌的PCR分析结果 1.尖孢镰刀菌;2.木贼镰刀菌;3.毁灭柱孢菌;4.厚垣镰刀菌;5.草茎点霉;6.链格孢;7.茄腐镰刀菌;8.弯孢霉;9.去离子水。

Fig.1 Result of PCR analysis of transmembrane-like protein-specific primers for detection Fusarium oxysporum 1.Fusarium oxysporum;2.F.equiseti;3.Cylindrocarpon destructans;4.F.chlamydosporum;5.Phoma herbarum;6.Alternaria alternata;7.F.solani;8.Curvularia;9.Sterilized ddH₂O.

图2 LAMP反应特异性扩增曲线

Fig.2 Amplification curves of LAMP specificity

图3 LAMP体系优化扩增曲线图 A.不同引物浓度的扩增曲线;B.不同温度条件下的扩增曲线;C.不同浓度Mg²⁺的扩增曲线;D.不同浓度dNTP的扩增曲线。

Fig.3 LAMP system optimization amplification curve A.Amplification curves at different primer concentration;B.Amplification curves at different temperatures; C.Amplification curves at different concentration of Mg²⁺;D.Amplification curves at different concentration of dNTP.

图4 LAMP反应灵敏度分析的扩增曲线和标准曲线 A.LAMP扩增曲线:1~6.0.02 pg/μL~20 ng/μL梯度稀释的pGEM-T-TP质粒;B.LAMP标准曲线的建立。

Fig.4 Amplification curves and standard curve for analysis of LAMP sensitivity A.LAMP amplification curves:1-6.The gradient dilution of pGEM-T-TP plasmids with concentration of 0.02 pg/μL-20 ng/μL;B.Establishment of LAMP standard curve.

图5 LAMP检测三七及土壤样品中的尖孢镰刀菌

Fig.5 LAMP detection of F.oxysporum in P.notoginseng and soil samples

图6 LAMP可视化检测三七及其土壤样本中的尖孢镰刀菌

Fig.6 Visual inspection of F.oxysporum in P.notoginseng and soil samples through LAMP

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