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Gene chip for detecting plant virus

A technology of gene chip and plant virus, applied in the field of plant virus detection gene chip

Inactive Publication Date: 2005-03-30
北京金长河科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many theoretical and application achievements in the detection of gene chips, but they are mainly focused on the diagnosis of human diseases, etc., and the use of gene chips in the detection of plant viruses has not been reported yet.

Method used

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  • Gene chip for detecting plant virus
  • Gene chip for detecting plant virus
  • Gene chip for detecting plant virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1: the preparation of chip

[0066]AlMV, CMV, PVY, PVA, PVX, PMTV, PLRV, TMV, PVM, BBTV, LSV, SqMV, CEVD, ASGV, PXP, PVS, LMoV, DsMV, ZYMV, WMV, CRSV, CCCVd, PNRSV, SBMV, TRSV, ComDV, AbYV, ComMV, AmLMV, CGVBV, ArjMV, CMABMV, ArLV, CpRMV, AV-1, CriMV, BaMMV, DVY, BaYMV, BaMMoV, DDMV, BBrMV, DNV, BCMNV, DSTV, BCMV, DesMV, BtMV, DAV, BiMV, DGBMV, BioMoV, EGMV, CdMV, FreMV, CVMV, GEV, CTLV, GSLV, CBSV, GGMV, CasYSV, HVY, CeMV, HMV, CpBDV, HiMV, ClYVV, HyaMV, CDV, IPMV, IFMV, TVBMV, IMMV, TVMV, ISMV, TWV, JGMV, ToPV, KVY, TZV, LYSV, TrMV, LMV, TmMV, LiMoV, TV-1, MDMV, TV-2, MVCV, TuV, MaMoV, TBBV, NDV, TBV, PPV, TCBV, PkMV, TuMV, UMV, PVV, ValMV, VanMV, PrMV, VDMV, RanMV, WMV-1, SrMV, WMV-2, SMV, WPMV, SVY, WVMV, Genomic nucleotide sequences of SCMV, YMV, SPFMV, ZYFV, SPVG, TamMV, TeMV, TEV and viroid PSTVD, especially the 3′ end sequences, were analyzed for conservative sequences, so as to design 2 to 5 probes for each virus , and synthesize the DNA. Dilute ...

Embodiment 2

[0067] Embodiment 2: utilize gene chip to detect plant virus

[0068] 1. Sampling: take the stem and leaf tissue of potato seed potato tubers planted under greenhouse isolation conditions after accelerated germination.

[0069] 2. Extraction of total RNA from the sample: Take 0.2g sample, extract its total RNA by conventional methods, and use it as a template for RT-PCR.

[0070] wxya 2 o

34.5μl

10×PCR buffer

5.0μl

d(ATG)TP(10mM)

1.0μl

dCTP (1mM)

1.0μl

Cy5-dCTP (1mM)

1.0μl

Primer L & R (5μM)

3.0μl

template

4.0μl

Taq polymerase

0.5μl

total capacity

50.0μl

[0071] The reaction mixture was centrifuged and mixed at a low speed, and PCR amplification was carried out. The amplification reaction was 35 cycles, and the extension reaction was 10 minutes at 72°C. Take 8 μl for agarose gel electrophoresis to detect the amplification effect.

[0072] 4. Hybridization: Precipit...

Embodiment 3

[0076] Embodiment 3: Utilize random primer to carry out gene chip to detect plant virus

[0077] The total RNA extraction of the sample to be tested was the same as in Example 2. Take 5 μl of total RNA (about 50 ng / μl) as a template for the synthesis of the first-strand cDNA, and synthesize the first-strand cDNA fragment according to the conventional RT method. The cDNA was recovered by ethanol precipitation, and Cy5 labeling under PCR conditions was carried out in the PCR reaction system with a random primer (Takara Code NO.D3802) containing 9 nucleotides, and then chip hybridization and detection were performed. The reaction conditions were the same as in the examples 2. Two sample preparation repetitions are set for the gene chip detection. As a result, the positive rate was 100%.

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PUM

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Abstract

This invention relates to a gene-chip for plant virus detection, particularly it relates to a method, by utilizing gene-chip, to detct samples if it contains plant virus. It is used for novel nucleic acid primer of PCR, or, for detecting PCR product probe. This method can be used for detecting simultaneously determinand sample with 2-122 kinds of virus, sich as: AIMV, CMV, PVA, PVX, PVY, TMY, BBTV, LSV, SqMV, CEVD, ASGV, PXP, PVS, LMoV, DsMV, ZYMV, WMV, CRSV, CCCVd, PNRSV, SBMV, TRSV and viroid PSTVd etc.

Description

(1) Technical field [0001] The invention relates to a gene chip for detecting plant virus, more specifically to a method for detecting whether plant virus is contained in a sample by using the gene chip, new nucleic acid primers for PCR and probes for detecting PCR products. (2) Background technology [0002] Plant virus is a ubiquitous disease factor. Plant virus infection brings serious economic losses to agriculture and is also one of the bottleneck factors for sustainable agricultural development. At present, the annual direct loss of crops caused by plant viruses in the world is more than 20 billion U.S. dollars, of which the losses to China are as high as tens of billions of RMB, which does not include local unique economic crops, wild and semi-wild resource plants, and Efforts and costs made to control viral diseases do not include indirect losses caused by plant viruses causing crop quality degradation. my country's crop planting, horticulture, and medicinal materia...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈集双杜志游郎秋蕾王冲陈洁云刘文洪谷庆琪
Owner 北京金长河科技发展有限公司
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