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Barley yellow dwarf virus specific primer, probe preparation and rapid detection method

A technology for barley yellow dwarf virus and detection method, which can be applied in the directions of biochemical equipment and methods, determination/inspection of microorganisms, etc., and can solve problems such as poor specificity and the like

Inactive Publication Date: 2006-10-25
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the specificity of its detection is poor, and false positive reactions often occur

Method used

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  • Barley yellow dwarf virus specific primer, probe preparation and rapid detection method
  • Barley yellow dwarf virus specific primer, probe preparation and rapid detection method
  • Barley yellow dwarf virus specific primer, probe preparation and rapid detection method

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Effect test

Embodiment 1

[0051] The preparation and detection method of the specific primer of embodiment 1.BYDV-GPV strain, the cDNA probe of digoxigenin labeling

[0052] 1.1 Design of specific primers

[0053] Use Vector NT software to carry out primer design, determine the primer (numbering SEQ1, SEQ2) of BYDV-GPV probe as: (object fragment size is about 1300bp)

[0054] Upstream primer: 5'ATG AGT ACG GTC GCC 3'15bp

[0055] Downstream primer: 5'TTC GTC AAG CGT AAC TGT 3'18bp

[0056] 1.2 Extraction of RNA

[0057] Take 0.1g of BYDV-GPV, GAV and PAV poison source and healthy plant leaf tissue, add liquid nitrogen to fully grind, then add 1ml TRIZOL reagent (purchased from Invitrogen Company) to fully grind, leave at room temperature for 5min; add 200μl of chloroform, fully shake for 15sec , place at room temperature for 2-3min; 12,000rpm, centrifuge at 4°C for 10min; take the upper aqueous phase into a new tube, add 0.5ml isopropanol, and place at room temperature for 10min; 1,2000rpm, centrifu...

Embodiment 2

[0073] Preparation and detection method of the specific primer of embodiment 2.BYDV-GAV strain, the cDNA probe of digoxigenin labeling

[0074] 2.1 Design of specific primers

[0075] Use Vector NT software to carry out primer design, determine the primer (numbering SEQ 3, SEQ4) of BYDV-GAV probe as: (object fragment size is about 960bp)

[0076] Upstream primer: 5'ATG AAT TCA GTA GGC CGT AGA A3' (22bp)

[0077] Downstream primer: 5'GTC TCG GTT TCC TCC AAT GTG 3' (21bp)

[0078] 2.2 Extraction of RNA

[0079] The operation is the same as 1.2.

[0080] 2.3 Optimization of RT-PCR conditions

[0081] 2.3.1 Synthesis of cDNA

[0082] In the PCR reaction tube, add RNA extraction solution 1ul; downstream primer (50pmol / ul) 1ul; 5×MMLVFirst strand Buffer 4ul; ddH 2 O 4ul; after mixing, denature at 90°C for 1 min, and place on ice for 2 min. Then add the following reagents in turn: dNTPs (10mmol / L) 2ul; Rnasin (40U / ul) 1ul; DTT (100mM) 1ul; MgCl 2 (25mM)1ul; MMLV Reverse Transcr...

Embodiment 3

[0096] The preparation and detection method of the specific primer of embodiment 3.BYDV-PAV strain, the cDNA probe of digoxigenin labeling

[0097] 3.1 Design of specific primers

[0098] Use VectorNT software to carry out primer design, and determine the primers (numbered as SEQ5, SEQ6) of BYDV-PAV probe as: (the target fragment size is about 1500bp)

[0099] Upstream primer: 5'GTA CAA GGC AAA TGG CAC GAC 3'

[0100] Downstream primer: 5'GTT CTG CCT GTT TCC CAG CAT 3'

[0101] 3.2 Extraction of RNA

[0102] The operation is the same as 1.2.

[0103] 3.3 Optimization of RT-PCR conditions

[0104] 3.3.1 Synthesis of cDNA

[0105] In the PCR reaction tube, add 1ul of viral RNA extraction solution; 1ul of downstream primer (50pmol / ul); 4ul of 5×MMLV First strand Buffer; ddH 2 O 4ul; after mixing, denature at 90°C for 1 min, and place on ice for 2 min. Then add the following reagents in turn: dNTPs (10mmol / L) 2ul; Rnasin (40U / ul) 1ul; DTT (100mM) 1ul; MgCl 2 (25mM)1ul; MML...

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Abstract

"Specific primers, probe preparation and rapid detection method for barley yellow dwarf virus" of the present invention. The specific primer of barley yellow dwarf virus is characterized in that barley yellow dwarf virus GPV strain, specific primer is SEQ 1, SEQ 2; Barley yellow dwarf virus GAV strain, specific primer is SEQ 3, SEQ 4; Barley yellow dwarf virus Dwarf virus PAV strain, specific primers are SEQ5, SEQ6. The present invention uses my country's barley yellow dwarf virus GPV, GAV and PAV three kinds of strains as materials, adopts its specific primer, uses non-radioactive substance digoxin as a marker, and effectively prepares barley yellow dwarf virus GPV, GAV and PAV by PCR method. The cDNA probes of three strains of GAV and PAV, and the establishment of barley yellow dwarf virus nucleic acid dot hybridization detection technology, provide a simple, effective and safe technical method for the specific detection of barley yellow dwarf virus strains .

Description

Technical field: [0001] The invention belongs to the field of biotechnology. Further, the present invention relates to a method for detecting different strains of barley yellow dwarf virus that causes wheat yellow dwarf disease. Background technique: [0002] Wheat yellow dwarf disease caused by Barley yellow dwarf virus (BYDV) is one of the most widely distributed and most serious cereal virus diseases in the world. my country's Northwest, North China, Northeast China, Central China, Southwest China and East China wheat areas are distributed, of which Shaanxi, Gansu, Ningxia, Inner Mongolia, Shandong, Hebei, Henan, Shanxi and other provinces are the main endemic areas. Since the 1960s, seven large-scale epidemics have caused disasters in my country, and the average yield of the affected wheat has been reduced by about 40%, and the severity can reach more than 70%, so that the harvest has failed. In 1970 and 1987, Shaanxi and Gansu alone lost more than 900 million kilogram...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 刘艳王锡锋周广和李莉
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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