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A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant

A technology of nucleotide sequences and leguminous plants, applied in the measurement/testing of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of laborious detection cycle, time-consuming, lack of detection methods for pathogenic organisms, etc.

Inactive Publication Date: 2007-04-25
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU +3
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many shortcomings in these methods: 1. Time-consuming, laborious and long detection cycle, which does not conform to the spirit of "speeding up customs clearance" proposed by the State Council; 2. Non-specific reactions are common in the detection process; 3. Some pathogenic organisms (especially plant viruses) lack of effective detection methods
None of these can meet the needs of my country's rapid economic development

Method used

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  • A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant
  • A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant
  • A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1, preparation gene chip

[0100] 1. Materials and methods

[0101] 1. Materials

[0102] Centrifuge: purchased from Heraeus.

[0103] PCR instrument: purchased from Techne Company.

[0104] Spotting instrument: purchased from GeneMachines, model: OminiGrid.

[0105] Chip assembly tools: purchased from Beijing Boao Biochip Co., Ltd.

[0106] Hybridization box: purchased from Beijing Boao Biochip Co., Ltd.

[0107] Chip scanner: purchased from Axon Company, model: GenePix4100A.

[0108] Bioinformatics and chip analysis software: free version or trial version, ClustalX1.83, VectorNTI9.1, ArrayDesigner2.02, GenePix Pro4.0 (purchased from Axon).

[0109] Probes, primers, and labeled control templates: see Table 1-Table 11, synthesized at Beijing Aoke Biotechnology Co., Ltd.

[0110] Blank control: 50% DMSO, purchased from Sigma.

[0111] Film substrate: Aldehyde glass substrate, purchased from CEL Company and Beijing Boao Biochip Co., Ltd.

[0112] Spott...

Embodiment 2

[0137] Embodiment 2, the using method of gene chip

[0138] 1. Extraction of total RNA from the sample to be tested (recommended method):

[0139] 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;

[0140] Centrifuge at 12000g (about 12000rpm) at 4°C for 10min, and transfer the supernatant to a new 1.5mL centrifuge tube;

[0141] 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;

[0142] 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;

[0143] Centrifuge at 12000g (about 12000rpm) at 4°C for 10min, discard the supernatant, add 1mL of 75% ethanol (DEPC wate...

Embodiment 3

[0195] Embodiment 3, chip detection sensitivity test

[0196] 1. Method:

[0197] 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.

[0198] 2. Results:

[0199] 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.

[0200] 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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Abstract

A gene chip for detecting viruses of leguminous plant is disclosed, several probes and nature-control collations are fixed on surface of solidoid carrier, the probes has the nucleotide sequence showed on from SEQ ID No.1 to SEQ ID No.80; the nature-control collations consists of the surface chemistry LUT, fluorescence labeling LUT, positive LUT, negative LUT, cross LUT and blank control. The advantages and innovations of the invention are following: practicability, quick-speed, and idiocrasy, and sensitivity, low activity cost, repeatability, stable and reliable results. The testing system can guarantee the repeatability and accuracy of results that is proofed by the repeatability and stability test, the system can be widely used in area of plant quarantine of the outbound and enter the country test and quarantine system, the safety supervision of plant source food, the diagnosis of plant virus in plant protecting station and the authenticate of pathogenesis in research of plant taxonomic virology. The gene chip testing call for detecting viruses of leguminous plant can be used in about 600 daily quarantine pursuits of leguminous plant source food.

Description

technical field [0001] The invention relates to a gene chip, nucleotide sequence and kit for detecting leguminous plant viruses, belonging to the field of inspection and quarantine. Background technique [0002] The issue of food safety is a historical issue, and it is also a global issue. Historically, due to the lack of necessary detection methods, pathogenic organisms were introduced from one place to another, causing the spread of pathogenic organisms in the introduced place, causing huge impact and loss on local human survival, health and agricultural production safety. . For example, the famous "Irish famine" was caused by the introduction of potatoes from the Americas, which caused the epidemic of potato late blight. In Ireland, which has a population of only 8 million, more than 200,000 people died of famine, and 1.64 million people fled due to famine. [0003] In many countries in the world, there have been safety problems of plant-derived food caused by pathogeni...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12Q1/68
Inventor 汪琳吴小兵相宁周雪平周琦任鲁风马新颖高文娜李明福邓丛良王欣月张永江汪万春种焱王甲正何新舟于翠吴建祥
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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