Method for detecting gene F200Y of fusarium graminearum resisting carbendazim on basis of loop-mediated isothermal amplification technology and primer composition
A technology of Fusarium graminearum and primer composition, applied in the biological field, can solve the problems of inability to quickly detect carbendazim-resistant strains of Fusarium graminearum, expensive thermal cycle instruments, low accuracy, etc., and achieve the goal of getting rid of heat Reliance on the cycler, saving equipment, good practical effect
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Embodiment 1
[0034] Embodiment 1 Specificity experiment of LAMP reaction primer composition
[0035] In order to verify the specificity of the LAMP reaction primer composition, Fusarium graminearum β 2 Based on the mutation of the 200th amino acid codon TTC(Phe)→TAC(Tyr) of tubulin (FGSG_06611.3), LAMP primers were designed, and the mutation site was located at the 3' end of the forward internal primer FIP. Any or any two bases were mismatched between the 3 bases upstream and downstream of the point for mismatch mutation, and the DNA of the sensitive strain of Fusarium graminearum and the carbendazim-resistant genotype F167Y strain of Fusarium graminearum were used as LAMP experiment was carried out on the template, and a LAMP primer composition capable of specifically identifying the carbendazim-resistant genotype F200Y strain of Fusarium graminearum was optimized.
[0036] The specific sequences of each primer are as follows:
[0037] FIP: 5'-GGCGATCTTGAGGGTCCTCTCGAGAACTCTGACGAGAC AC...
Embodiment 2
[0042] Example 2 LAMP reaction system and detection kit system optimization
[0043] In order to save the identification cost and ensure the stability and reliability of the identification method, BstDNA polymerase (8U / μL) (0.8-4.0U), Mg2+ (25mM) concentration (0.6-2.0μL), primer FIP / BIP (40μM) and F3 / B3 (10μM) concentration (0.1-0.5μL), betaine (8M) concentration (0.4-2.0μL), HNB (2.5mM ) concentration (0.4-1.2μL) was optimized, and the best system (1mL detection solution) of each component in the kit was determined to be: 320U Bst DNA polymerase, 20mM Tris-HCl, 10mM KCl, 10mM (NH4) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 2.5mM MgCl 2 , 0.8mM dNTPs, 0.96M Betaine, 0.2mM Hydroxybromophenol Blue (HNB), 1.0μM Forward Inner Primer FIP, 1.0μM Reverse Inner Primer BIP, 0.25μM Forward Outer Primer F3, 0.25μM Reverse Outer Primer B3, prepared by adding sterile ultrapure water, packaged 100 times, transported at low temperature, stored at -20°C, and valid for 1 year.
[0044] Whe...
Embodiment 3
[0049] Embodiment 3 LAMP reaction parameter optimization
[0050] In order to obtain the most suitable reaction temperature and time and ensure the high efficiency of the detection method, the reaction temperature and time in the reaction parameters were optimized. Able to achieve LAMP amplification.
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