RNA-RP RT-PCR method capable of fast detecting melon viruses
A technology for melons and viruses, applied in the directions of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., to achieve the effect of saving cost and time, and simple operation
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Embodiment 1
[0050] The screening of solution used in the quick release of embodiment 1 plant total RNA
[0051] Weighed 8 copies of 0.05 g of CGMMV melon diseased leaves, put them in 2 mL centrifuge tubes, and crushed them with liquid nitrogen at low temperature using a tissue disruptor, and added 1 mL of RNase-free ddH2O, 1×PBS, PBST, PBST+2%PVP, 0.5% Triton X-100 aqueous solution, 0.5% Triton X-100 PBS solution, 0.5% Triton X-405 aqueous solution and 0.5% Triton X-405 PBS solution, shake and mix vigorously, 8000 rpm Centrifuge for 5 min, absorb the supernatant for later use;
[0052] Use the CGMMV-specific primer sequence CG-3467F / CG-5294R to carry out virus detection on the prepared supernatant, in which 2×1 Step Buffer (Dye PLus) 10 μL, PrimeScript 1 Step Enzyme Mix 0.8 μL, CG-3467F (10 μM) 1 μL, CG-5294R (10 μM) 1 μL, supernatant 0.5 μL, rehydration 6.7 μL, total PCR system 20 μL; 50°C for 30 min, 94°C for 2 min, 94°C for 30 sec, 57°C for 30 sec, 72°C 2 min, run 35 cycles, total ex...
experiment example 2
[0055] Experimental example 2 RNA-RP RT-PCR detection of CGMMV in tobacco and cucumber
[0056] Weigh two copies of 0.05 g of CGMMV tobacco leaves and cucumber diseased leaves, put them in 2 mL centrifuge tubes, crush them with liquid nitrogen at low temperature with a tissue breaker, and add 1 mL of PBST+2% to each tube sample PVP and 0.5% Triton X-405PBS solution, shake vigorously, mix well, centrifuge at 8000 rpm for 5 min, absorb the supernatant, and use the CGMMV-specific primer sequence CG-3467F / CG-5294R to carry out virus detection on the prepared supernatant, wherein 2×1 Step Buffer (Dye PLus) 10 μL, PrimeScript 1 Step Enzyme Mix 0.8 μL, CG-3467F (10 μM) 1 μL, CG-5294R (10 μM) 1 μL, supernatant 0.5 μL, water 6.7 μL, total A total of 20 μL of PCR system; 35 cycles of 30 min at 50°C, 2 min at 94°C, 30 sec at 94°C, 30 sec at 57°C, 2 min at 72°C, and a total extension of 2 min at 72°C;
[0057] 4 μL of PCR products were taken for electrophoresis detection, and the results...
experiment example 3
[0058] Experimental example 3 RNA-RP RT-PCR detection of CMV in tobacco and melon
[0059] Weigh 0.05 g of CMV benthamiana benthamiana and melon diseased leaves respectively, put them in 2mL centrifuge tubes, crush them with liquid nitrogen at low temperature with a tissue breaker, and add 1 mL of PBST+2%PVP to each tube sample and 0.5% Triton X-405PBS solution, shake vigorously, mix well, centrifuge at 7000 rpm for 8min, absorb the supernatant, and use specific primers CMV-R1-1F / CMV-R1-1627R combined with a one-step RT-PCR method to detect CMV, Concrete step is the same as embodiment 1, is not repeating,
[0060] 3 μL of PCR products were taken for electrophoresis detection. The results are shown in 5, indicating that both PBST+2%PVP and 0.5% Triton X-405PBS solutions can effectively release the total RNA in Nicotiana benthamiana and melon leaves for the detection of CMV.
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