PCR (Polymerase Chain Reaction) detection reagent and kit of Japanese pratylenchus coffeae
A detection kit and detection reagent technology, which are applied in the field of PCR detection reagents and kits of Brachybody japonica, can solve the problems of unpublished molecular biology detection reports of Brachybody japonica, and achieve strong practicability and high sensitivity Effect
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Embodiment 1
[0012] Embodiment 1, the PCR detection kit of Brachybody nematode japonicus
[0013] 100 or 200 reaction system, 50μL reaction system includes: Mg-free 2+ 5.0 μl of 10x PCR buffer with a concentration of 25 mM MgCl 2 5.0 μL solution, 4.0 μL dNTP solution with a concentration of 0.1 mM, 0.6 μL Taq enzyme solution with a concentration of 5 U / μL, 3.0 μL each of upstream primer solution and downstream primer solution with a concentration of 10 μM. Taq enzyme solution and dNTP solution were purchased from Treasure Bioengineering (Dalian) Co., Ltd., wherein the upstream primer solution contained a primer with the nucleotide sequence GGCAACGGGC CTAGTTATGA, and the downstream primer solution contained a primer with the nucleotide sequence GACTCGCGCACACGATAAAC.
Embodiment 2
[0014] Embodiment 2, DNA extraction method
[0015] Pick a single nematode and put it into a 200 μL PCR tube [add 10 μL double distilled water and 5 μL 10×PCR buffer (without Mg 2+ )], placed in liquid nitrogen for more than 1 min (or -70 °C for 0.5 h), and immediately heated at 85 °C for 2 min after taking it out. Then add 1 μL of 1 mg / mL proteinase K to the PCR tube, heat at 56°C for more than 30 minutes, and heat at 95°C for 10 minutes to obtain a DNA template.
Embodiment 3
[0016] Embodiment 3, PCR detection method
[0017] Use the PCR detection kit of Brachybody japonica for detection, and the PCR reaction system is: no Mg 2+ 5.0 μl of 10x PCR buffer with a concentration of 25 mM MgCl 2 5.0 μL solution, 4.0 μL dNTP solution with a concentration of 0.1 mM, 0.6 μL Taq enzyme solution with a concentration of 5 U / μL, 3.0 μL each of upstream primer solution and downstream primer solution with a concentration of 10 μM, DNA template 1.0-5.0 μL, ddH 2 O make up 50 μL. PCR reaction conditions: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30S, annealing at 55°C for 30S, extension at 72°C for 30S, a total of 35 cycles; after the last cycle, extension at 72°C for 6min. Electrophoresis: 5 μL of PCR products were separated by 1.0% (weight / volume) agarose gel electrophoresis, stained with DNA dyes, and visualized under an ultraviolet gel imaging system. If the specific fragment size was 176bp, it was Brachybody japonicus .
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