Loop-mediated isothermal amplification assay for detection of Sclerotinia sclerotiorum in sunflower
A color change-based sensitive detection method was developed using loop-mediated isothermal amplification(LAMP). The LAMP specific primers were selected for the detection of Sclerotinia sclerotiorum. The LAMP reaction system and optimal conditions were established,followed by sensitivity and specificity tests. The re-sults demonstrated that F3-1:ATGCCTGTTCGAGCGTCA,B3-1:AGTTCAGCGGGTATCCCTA,FIP-1:GCCGC-CACTGATTTTAGAGCCTTTTCAACCCTCAAGCTCAGC and BIP-1:TCGTTACAGGTTCTCGGTGTGCCCT-GATCCGAGGTCAACCAT were used as primers. The reaction system comprised 10×Bst Reaction Buffer 2.4 μL,dNTP Mixture 1.28 mmol·L-1,F3/B30.15 μmol·L-1,FIP/BIP 0.8 μmol·L-1,HNB 150 μmol·L-1,MgSO41.2 mmol·L-1,Bst DNA polymerase 0.19 U·L-1 and ddH2O 13.4 μL. Nucleic acid amplification reaction was performed at 64℃ isothermal conditions for 1h. The test results can be visually assessed with the naked eye,while agarose gel electrophoresis verification showsd clear trapezoidal amplification results. These phenomena were not observed in other tested strains such as Botrytis cinerea,phytophthora,saprophyticus,etc.,indicating a strong specificity to S. sclerotinia. The minimum detection limit of LAMP technology was determined to be 1×10-3 ng·μL-1. The establish-ment of this method provides a novel technique for the quarantine and rapid diagnosis of the diseases caused by S. sclerotiorum.
Sclerotinia sclerotiorumloop-mediated isothermal amplification techniquehydroxynaphthol blue (HNB)
万方数据
维普
