Establishment and Application of Real-time Fluorescent LAMP Rapid Detection of Fusarium oxysporum of Panax notoginseng Root Rot

doi:10.7668/hbnxb.20194307

Abstract

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

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.

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Figures/Tables 6

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.

Fig.2 Amplification curves of LAMP specificity

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.

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.

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

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

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