A gene chip, nucleotide sequence and reagent kit for detecting virus of potato family plant
A nucleotide sequence, plant virus technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve non-compliance, lack of detection methods for pathogenic organisms, and cannot meet the needs of rapid economic development And other issues
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Embodiment 1
[0098] Embodiment 1, preparation gene chip
[0099] 1. Materials and methods
[0100] 1. Materials
[0101] Centrifuge: purchased from Heraeus.
[0102] PCR instrument: purchased from Techne Company.
[0103] Spotting instrument: purchased from GeneMachines, model: OminiGrid.
[0104] Chip assembly tools: purchased from Beijing Boao Biochip Co., Ltd.
[0105] Hybridization box: purchased from Beijing Boao Biochip Co., Ltd.
[0106] Chip scanner: purchased from Axon Company, model: GenePix4100A.
[0107] Bioinformatics and chip analysis software: free version or trial version, ClustalX1.83, VectorNTI9.1, ArrayDesigner2.02, GenePix Pro4.0 (purchased from Axon).
[0108]Probes, primers, and labeled control templates: see Table 1-Table 11, synthesized at Beijing Aoke Biotechnology Co., Ltd.
[0109] Blank control: 50% DMSO, purchased from Sigma.
[0110] Film substrate: Aldehyde glass substrate, purchased from CEL Company and Beijing Boao Biochip Co., Ltd.
[0111] Spotti...
Embodiment 2
[0136] Embodiment 2, the using method of gene chip
[0137] 1. Extraction of total RNA from the sample to be tested (recommended method):
[0138] Take 0.1g of the sample to be tested, cut it into small pieces, grind it into powder with liquid nitrogen, add 1mL Trizol reagent to continue grinding and homogenate, transfer it into a sterilized 1.5mL centrifuge tube and let it stand at room temperature for 5min;
[0139] Centrifuge at 12000g (about 12000rpm) at 4°C for 10min, and transfer the supernatant to a new 1.5mL centrifuge tube;
[0140] Add 0.2mL chloroform, shake vigorously for 15s, then keep at room temperature for 2-3min, centrifuge at 12000g (about 12000rpm) for 15min at 4°C;
[0141] Transfer the upper aqueous phase (about 0.6mL) to a new 1.5mL centrifuge tube, add 0.5mL isopropanol, invert and mix well, and keep at room temperature for 10min;
[0142] Centrifuge at 12000g (about 12000rpm) at 4°C for 10min, discard the supernatant, add 1mL of 75% ethanol (DEPC wate...
Embodiment 3
[0194] Embodiment 3, chip detection sensitivity test
[0195] 1. Method:
[0196] RNA was extracted from plant samples (provided by Beijing Entry-Exit Inspection and Quarantine Bureau) carrying tobacco ringspot virus (Tobacco ringspot virus, TRSV), and carried out 10 times respectively. 1 ~10 3 1-fold serial dilution, RT-PCR detection, ELISA (purchased from Beijing Sains Technology Co., Ltd.) and chip detection were performed respectively.
[0197] 2. Results:
[0198] The results of RNA dilution RT-PCR detection of TRSV samples are shown in Figure 3-1, where 1:10 -3 Dilution; 2:10 -4 Dilution; 3.10 -5 Dilution; n: blank control; M: Marker DL2000, only 10 -3 Dilution can detect the virus.
[0199] The ELISA test results of multiple dilutions of the TRSV sample grinding solution are shown in Figure 3-2. When the TRSV sample grinding solution is diluted 1:4, the positive signal value is close to the critical value, and when the dilution is 1:8, no positive signal can be j...
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