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. 2017 Jun 26;7(1):4253.
doi: 10.1038/s41598-017-04084-y.

Novel loop-mediated isothermal amplification (LAMP) assay with a universal QProbe can detect SNPs determining races in plant pathogenic fungi

Yu Ayukawa  1   2 , Saeri Hanyuda  2 , Naoko Fujita  2 , Ken Komatsu  3   4 , Tsutomu Arie  1   2
Affiliations

Novel loop-mediated isothermal amplification (LAMP) assay with a universal QProbe can detect SNPs determining races in plant pathogenic fungi

Yu Ayukawa et al. Sci Rep. .

Abstract

Tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici (Fol) is grouped into three races based on their pathogenicity to different host cultivars. Rapid detection and discrimination of Fol races in field soils is important to prevent tomato wilt disease. Although five types of point mutations in secreted in xylem 3 (SIX3) gene, which are characteristic of race 3, have been reported as a molecular marker for the race, detection of these point mutations is laborious. The aim of this study is to develop a rapid and accurate method for the detection of point mutations in SIX3 of Fol. Loop-mediated isothermal amplification (LAMP) of SIX3 gene with the universal QProbe as well as two joint DNAs followed by annealing curve analysis allowed us to specifically detect Fol and discriminate race 3 among other races in about one hour. Our developed method is applicable for detection of races of other plant pathogenic fungi as well as their pesticide-resistant mutants that arise through point mutations in a particular gene.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Design of LAMP primers and joint DNAs Partial SIX3 nucleotide sequences (positions 59 to 282) used for designing LAMP primers and joint DNAs. Positions of the designed primers and joint DNAs are indicated by arrows. Point mutation sites are shown by an open box.
Figure 2
Figure 2
Optimization of LAMP reaction temperature with the designed SIX3 primer sets. LAMP was performed with DNA of Fol race 1 at different temperatures from 59 to 66 °C for 20 min. Similar results were obtained in two independent experiments.
Figure 3
Figure 3
Melting temperature of joint DNAs combined with the universal QProbe. Melting curve analysis with Joint DNA 1 (a) or Joint DNA 2 (b) was performed from 35 to 90 °C following 60 min of LAMP reactions using genomic DNAs of Fol isolates of different races as a template. Similar results were obtained in two independent experiments.
Figure 4
Figure 4
Comparison of relative sensitivity of LAMP with the universal QProbe and conventional PCR (a) Melting curve analysis following LAMP reaction with serial tenfold dilutions of genomic DNA of Fol race 1 (3 ng to 3 fg) using Joint DNA 1. (b) Agarose gel electrophoresis of PCR products amplified from the same dilutions of genomic DNA with SIX3-F3 and SIX3-B3 primers.
Figure 5
Figure 5
Typing of Fol races in artificially infested soil by LAMP using the universal QProbe with joint DNAs. Melting curve analysis of LAMP products using the QProbe combined with Joint DNA 1 (a) and Joint DNA 2 (b). Genomic DNAs of Fol or artificial infested soil DNAs by Fol were used as a template. Similar results were obtained in two independent experiments.
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References

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