Fluorescent quantitative PCR (polymerase chain reaction) detection method for quickly detecting Botrytis cinerea and kit
A real-time fluorescence quantitative technology for Botrytis cinerea, applied in the field of crop disease diagnosis and control, can solve the problems of few applications, achieve high detection sensitivity, good specificity, and improve accuracy
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Embodiment 1
[0043] The specific primer detection of embodiment 1 tomato cinerea
[0044] 1) DNA preparation of each strain:
[0045] Cultivate strains on a plate, place them in an incubator at a suitable temperature, and grow for about a week. Scrape mycelia with a sterilized dissecting needle, collect them in EP tubes, and store them at -20°C for later use. The mycelium was ground with liquid nitrogen, and the DNA of each strain was extracted with Takara DNA extraction kit.
[0046] 2) Specific primers for detecting Botrytis cinerea:
[0047] Specific primer (CND11F): 5'-AACCCGACTTTGGACCTG-3'
[0048] Specific primer (CND11R): 5'-TTGCTTCCGATTGATTGC-3'
[0049] Synthesized by Shanghai Shenggong Company.
[0050] 3) PCR reaction system for detecting Botrytis cinerea:
[0051] PCR reaction system, in which l0×PCRbuffer 2μL, 15mM MgCl 2 0.5 μL, 1.6 μL of 2.5 mM dNTPs, 0.5 μL of each 5 μM primer, 0.2 μL of Taq enzyme (5U / μL), 1 μL of DNA template, 13.7 μL of sterile double-distilled wat...
Embodiment 2
[0059] The sensitivity detection of embodiment 2 tomato Botrytis cinerea specific primers
[0060] 1) Serial dilution of DNA template concentration of Botrytis cinerea from high to low:
[0061] The DNA template concentration of Botrytis cinerea was serially diluted from high to low by 10×, from a high concentration of 30 ng / mL to 0.003 ng / mL.
[0062] 2) Specific primers for detecting Botrytis cinerea:
[0063] Specific primer (CND11F): 5'-AACCCGACTTTGGACCTG-3'
[0064] Specific primer (CND11R): 5'-TTGCTTCCGATTGATTGC-3'
[0065] Synthesized by Shanghai Shenggong Company.
[0066] 3) PCR reaction system for detecting Botrytis cinerea:
[0067] PCR reaction system, in which l0×PCRbuffer 2μL, 15mM MgCl 2 0.5 μL, 2.5 mM dNTPs 1.6 μL, 5 μM primers 0.5 μL each, Taq enzyme (5U / μL) 0.2 μL, DNA template 1 μL, sterilized double distilled water 13.7 μL, the final volume is 20 μL.
[0068] 4) PCR amplification program for detecting Botrytis cinerea:
[0069] Pre-denaturation at 94...
Embodiment 3
[0074] Example 3 Fluorescence quantitative PCR method detects the quantity of Botrytis cinerea in diseased tomato plant leaves and fruits
[0075] 1) Sample DNA preparation:
[0076] The diseased leaves and fruits of different parts of tomato plants inoculated in the greenhouse were collected in EP tubes and stored at -20°C for later use. Each sample was ground with liquid nitrogen, and the DNA of the sample was extracted with the Takara DNA extraction kit.
[0077] 2) Specific primers for detecting Botrytis cinerea:
[0078] Specific primer (CND11F): 5'-AACCCGACTTTGGACCTG-3'
[0079] Specific primer (CND11R): 5'-TTGCTTCCGATTGATTGC-3'
[0080] Synthesized by Shanghai Shenggong Company.
[0081] 3) Fluorescent quantitative PCR reaction system for detecting Botrytis cinerea:
[0082] The total reaction volume is 20 μL, SYBRGreenSupermix 10 μL, specific primer CND11F (10 μM) 1 μL, specific primer CND11R (10 μM) 1 μL, DNA template 1 μL, and the balance is sterile double disti...
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