Abstract:

In order to develop a rapid and sensitive real-time fluorescent LAMP technique for quantitative detection of lotus rhizome rot pathogen (Fusarium commune), four LAMP specific primers were designed based on TEF-1α gene sequence unique to F. commune, a real-time fluorescent LAMP rapid detection method for pathogen of lotus rhizome rot was established, and the specificity, sensitivity and field application effects of this method were evaluated. The specific test showed that this method could identify and detect the lotus rhizome rot pathogen (F. commune) in 60 minutes under the constant temperature of 63℃. Among the 15 tested strains, 10 strains showed positive green and gradient bands in the electrophoretic test results, while the other 5 control strains showed orange and no gradient bands. The results of real-time fluorescent LAMP showed that the F. commune strains could expand normally with a single peak dissolution curve, while other control strains did not expand. Sensitivity experiments showed that the minimum detection limit of LAMP technology for F. commune was up to 100 fg/μL, and the minimum limit of soil strain content was 1×10³ spores per gram of soil. The standard curve of pathogen developed in this paper can be used for the quantitative detection of F. commune. This technique was also suitable for the detection of F. commune in lotus plants in the field before and after infection. The DNA content of F. commune in the underground rhizome tissue of lotus plants was positively correlated with the severity of the disease. The real-time fluorescent LAMP rapid detection technology established in this paper has the advantages of strong specificity, high sensitivity, simple and quick operation, which is suitable for the field rapid detection of F. commune.

Key words: lotus rhizome rots pathogen, TEF-1α gene sequence, real-time fluorescent, loop-mediated isothermal amplification, rapid detection, molecular testing, specific primers