Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

A technology of barley yellow dwarf virus and detection method, which is applied in the direction of biochemical equipment and method, microbial measurement/inspection, etc., and can solve the problems of poor specificity

Inactive Publication Date: 2005-12-21
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
View PDF0 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Examples

Experimental program
Comparison scheme
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 SEQ 1, 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 of isopropanol, place at room temperature for 10min 1,2000rpm, centrifug...

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 Transcrip...

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 Vector NT software to carry out primer design, and determine the primers (numbered as SEQ 5, 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; M...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to peculiarly primer of barley yellow dwarf, probe preparation and fast detection method. Its characteristic lies in: for GPV strain series, the peculiarly primers are SEQ1, SEQ2; for GAV strain series, the peculiarly primer as SEQ3, SEQ4; for PAVstrain series, the peculiarly primer as SEQ5, SEQ6. In the invention the non-radioactive material digoxin is used as the marking thing, and use PCR law to prepare the cDNA probe for the three kinds of GPV ,GAV and PAV effectively to provide a simple, safety and effective peculiarly measurement.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com