Quantitative detection method of tea-geometrid-type polyhedrosis viruses
A quantitative detection method and karyotype polyhedron technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc. Protect the interests of tea farmers, achieve full monitoring and high sensitivity
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Embodiment 1
[0027] The acquisition of embodiment 1, EoNPV virus sample and the extraction of genomic DNA thereof
[0028] Infect tea geometrid larvae with tea geometrid nuclear polyhedrosis virus (EoNPV), collect 100 poisoned worm carcasses, use PBS solution as buffer solution to homogenize repeatedly, the volume ratio of buffer solution to carcass is 10:1, centrifuge at 10000g for 10min , repeat 3 times, remove the precipitate, the supernatant is the crude virus extract; the crude virus extract is centrifuged at 35,000g for 12 hours, and the precipitate is the purified virus particle; the purified virus particle is washed repeatedly with 1×PBS until milky white up to; add 0.1mol / L Na 2 CO 3 , 0.05mol / L NaCl, 5 mL of lysate at pH 10.3, lyse polyhedrosis virion polyhedrosis virion of tea geometrid for 1-2h; centrifuge at 3000g for 5min, absorb supernatant, remove unlysed karyotype of tea geometrid Polyhedron virions; first extract with an equal volume of phenol: chloroform: isoamyl alcoh...
Embodiment 2
[0029] Embodiment 2, target fragment PCR amplification
[0030]According to the p10 gene of the EoNPV genome sequence (NC_008586.1), a pair of specific primers EoNPV p10F and EoNPVp10R were designed by using DNAStar software, which were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The primer sequences are: 5'-actgctctgcagcaacaagtcg-3 ' (see sequence listing SEQ No.1) and 5'-gtcgttgacggtggtgctaatc-3' (see sequence listing SEQ No.2), amplify p10, and use 25 μL system for PCR reaction (template 2 μL, 10×buffer 2.5 μL, dNTP 2 μL, 0.5 μL of forward and reverse primers, 0.25 μL of Tag enzyme, add sterilized double distilled water to 25 μL), PCR amplification uses a two-step method, the procedure is as follows: first 94 ° C for 3 min; then 94 ° C for 30 s, 62 ° C for 45 s; cycle 35 times, and finally extended at 72°C for 10min. Take 10ul of the PCR product and detect it by electrophoresis on a 1% agarose gel. For the test result, see figure 1 , which is consistent with th...
Embodiment 3
[0031] Embodiment 3, the preparation of plasmid standard and its concentration determination
[0032] The p10 gene fragment amplified in Example 2 was connected to the vector pMD18-T, transformed into Escherichia coli TG1, a single clone strain was picked, and the bacteria were shaken. Use the kit to extract single colony plasmid DNA and carry out PCR identification. The recombinant plasmids identified as positive were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Compare with the reference sequence to confirm the construction result. After the construction is completed, measure its concentration with a UV spectrophotometer, and dilute 1 μL of positive plasmid 100 times to measure its OD 260 and OD 280 value, calculate the recombinant plasmid concentration and OD 260 / OD 280 Ratio, the recombinant plasmid was diluted to 10 -10 copies / μL, stored in a -20°C refrigerator.
[0033] For the results of PCR identification of the recombinant plasmid, see figu...
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