DPO RT-PCR primer group, detection method and kit for synchronously detecting five citrus viruses and application of DPO RT-PCR primer group, detection method and kit
An OPORT-PCR, simultaneous detection technology, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of dead, chlorosis, frequent seedling transportation, etc., and achieve high sensitivity and accuracy. High precision, simple and efficient detection method
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Embodiment 1
[0034] Example 1 Primer Design
[0035] Based on the whole genome sequences of citrus decay virus, citrus leaf chipping virus, citrus split peel virus, citrus yellow veining virus, and citrus leaf mottle virus published by NCBI, DPO RT-PCR primers for simultaneous detection of these five viruses were designed.
[0036] Citrus decay virus (accession numbers: AF001623, AF260651, AY170468, FJ525436, JQ965169, etc.), citrus leaf fragmentation virus (accession numbers: AY646511, EU553489, MH108985, KC588948, KY706358, etc.), citrus split virus (accession numbers: DQ44444, etc.) M30870, S67437, M30868, etc.), all pathogenic types of citrus yellow veining virus (accession numbers: KT124646, KX156742, MK415924, MF563877, etc.), citrus leaf mottle virus (accession numbers: EU857539, MT863785, MG572236, MN495980, etc.) The whole genome sequence is compared and analyzed, and the conserved sequences between different pathogenic types of the same virus and the gene sequences with large dif...
Embodiment 2
[0050] Example 2 Construction of plasmid
[0051] Using the specific primers designed in Example 1, the target fragments were amplified from the collected samples of citrus and citrus decay virus, citrus leaf fragmentation virus, citrus split peel virus, citrus yellow veining virus, and citrus leaf mottle virus. The gel recovery kit recovered and purified the amplified fragment, ligated it with the pMDT-20T vector, and then transformed it into E. coli DH5α competent cells for cultivation, and carried out colony PCR verification to verify the correct colony, and sent the bacterial solution to Shanghai Biotechnology Co., Ltd. Engineering Bioengineering Co., Ltd. The colonies with the correct sequencing results were compared and the plasmids were extracted to obtain the recombinant plasmids pCEV, pCTV, pCLBV, pCTLV and pCYVCV. Then, the concentration of plasmid DNA was measured with a nucleic acid protein analyzer to determine the concentration of sample DNA, and the concentrati...
Embodiment 3
[0054] Example 3 Effects of different annealing temperatures on the detection results of single-plex PCR
[0055] Dilute 10 with cloned recombinant plasmid 2 times as a template for PCR amplification. The PCR reaction system was 20 μL: PremixEx TaqTM 10 μL, the final concentration of upstream and downstream primers was 0.50 μmol / L, the plasmid DNA template was 1 μL, and ddH2O supplemented 20 μL. The PCR reaction parameters were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30s, annealing at 48-62°C for 30s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 10min, cooling to 12°C to complete the reaction.
[0056] Other conditions remained unchanged, the annealing temperature was screened, and 8 treatments were set: 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, and 62.0°C.
[0057] PCR product electrophoresis detection: Mix 6μL of PCR product with 1μL of 6× loading buffer, and perform electrophoresis on a 1.2% agarose gel (with Goldview nucleic a...
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