Corn cyst nematode-specific scar markers, detection primers and rapid PCR detection method thereof
A cyst nematode and detection primer technology, which is applied in the field of corn cyst nematode-specific SCAR markers, detection primers and rapid PCR detection, can solve the problems of time-consuming morphological identification, heavy workload, and difficult application. To achieve the effect of short detection time, strong reliability and simple equipment requirements
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Embodiment 1
[0031] Determination of the specific SCAR markers of maize cyst nematode and the design of specific primers, including the following steps:
[0032] 1. Nematode DNA Extraction Reagent Preparation
[0033] (1) LB (Lysis Buffer) solution: 500mmol / L KCl, 10mmol / L Tris-HCl, 15mmol / LMgCl 2 , 1.0mmol / L DTT, 4.5% Tween20, sterilized at 121°C for 15min.
[0034] (2) Proteinase K: 600 μg / ml proteinase K, filter sterilized.
[0035] 2. DNA extraction
[0036] Pick a single corn cyst into a2 0.2mL centrifuge tube of O, frozen in liquid nitrogen, taken out and placed on ice, rotated in the centrifuge tube with a sterile glass rod until the ice melted, punctured the cysts, released eggs, added 8mL of LB solution, 2 mL of 600 mg / mL proteinase K solution, then frozen at -80°C for 30 min. The centrifuge tube was taken out, incubated at 65° C. for 90 min, and the supernatant was treated at 95° C. for 10 min, and the supernatant was used directly for subsequent PCR reaction as a nematode ...
Embodiment 2
[0056] The method of Example 1 was used to extract the DNA of different populations of Cyst Nematode and its related species Cyst Nematode, and the DNAs of the Cyst Nematode SCAR-specific molecular marker primers OPA03-HzF61 and OPA03-HzR456 developed above were used to carry out analysis on them respectively. PCR detection. The PCR amplification reaction system was 50 μL, and the PCR reaction system was 10×Buffer (containing Mg 2+ ) 5µL, 10mmol / L dNTP 4µL, primers OPA03-HzF1 and OPA03-HzR1 (10 µmol / L) 1µL each, Taq enzyme (5U / µL, Takara) 0.5µL, template DNA 5µL, sterilized ddH 2 O make up to 50 µL. PCR amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 45sec, annealing at 62°C for 30sec, extension at 72°C for 45sec; 35 cycles; extension at 72°C for 10min, and storage at 4°C. After PCR amplification, take 5 μL of amplification product and add 1 μL of loading buffer to electrophoresis on 1.5% agarose gel, stain with EB, observe and pho...
Embodiment 3
[0058] 1. Extraction of corn cyst nematode DNA
[0059] Pick a single corn cyst into a 2 0.2mL centrifuge tube of O, frozen in liquid nitrogen, taken out and placed on ice, rotated in the centrifuge tube with a sterile glass rod until the ice melted, punctured the cysts, released eggs, added 8mL of LB solution, 2 mL of 600 mg / mL proteinase K solution, then frozen at -80°C for 30 min. The centrifuge tube was taken out, incubated at 65°C for 90 minutes, and the supernatant was treated at 95°C for 10 minutes, and the supernatant was used as a nematode DNA template for subsequent PCR reactions.
[0060] 2. Amplification and Sequence Analysis of SCAR Fragments of Maize Cyst Nematode
[0061] The nematode DNA obtained in the previous step was diluted 10, 100, 1000 and 2000 times respectively as a template, and the SCAR marker primers OPA03-HzF61 and OPA03-HzR456 were used for amplification. The PCR amplification reaction system was 25 μL, and the PCR reaction system was 10×Buffer...
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