Real-time fluorescence PCR immobilization kit of wheat dwarf bunt germ (Tilletia controversa kuhn) and its detection method

A technology of wheat dwarf smut and real-time fluorescence, applied in biochemical equipment and methods, botany equipment and methods, organic chemistry, etc., can solve problems such as non-standardization, low-temperature storage, errors, etc., and achieve strong specificity and sensitivity High, short detection time effect

Inactive Publication Date: 2006-09-27
CHONGQING UNIV +2
View PDF0 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The real-time fluorescent PCR detection method is gradually being applied to the detection of plant pathogenic bacteria. However, the current detection kits cannot be stored at room temperature and need to be stored at low temperature, which causes incon

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
  • Real-time fluorescence PCR immobilization kit of wheat dwarf bunt germ (Tilletia controversa kuhn) and its detection method
  • Real-time fluorescence PCR immobilization kit of wheat dwarf bunt germ (Tilletia controversa kuhn) and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0056] Example one:

[0057] A real-time fluorescent PCR detection primer and immobilization kit based on SYBR Green I fluorescent dye, used for the specific detection of Tilletia dwarf, and its composition is:

[0058] [1] Sample nucleic acid extraction reagent: 3mol / L NaOH; TES buffer; 70% ethanol; nucleic acid eluate.

[0059] [2] Nucleic acid amplification solid-phase reagent:

[0060] The solid-phased mixture of real-time fluorescent PCR reaction reagents is in a dry gel form and can be used after adding water. The components include the following reagents: 1×PCR buffer, 2.6mmol / L MgCl 2 , 0.2mmol / L dNTP, 1Unit / 25uL Taq polymerase, each 0.5umol / L primer pair, biomacromolecule stabilizer to 23uL; 9umol / L SYBR Green I fluorescent dye.

[0061] The sequence of the oligonucleotide primer pair is: 5’-GAAGCTGGTGGAGGTG-3’

[0062] 5’-GACTGCCCAACGAAAA-3’ (serial number: NO.1);

[0063] [3] Harmless quantitative standard: the positive control freeze-dried prod...

Example Embodiment

[0065] Example two

[0066] A primer sequence based on real-time fluorescent PCR detection of Tilletia dwarf SYBR Green and a solid-phased kit, used for the specific detection of Tilletia dwarf, and its composition is:

[0067] Sample nucleic acid extraction reagents; nucleic acid amplification solid-phase reagents; harmless quantitative standards; testing supplies.

[0068] The oligonucleotide primer sequence is: 5’-TTTCGTTGGGCAGTCT-3’

[0069] 5'-ATCGGGTAAAGAAGCA-3' (serial number: NO. 2).

[0070] The sample nucleic acid extraction reagents, quantitative standards, and detection supplies are the same as in Example 1, and the components of the nucleic acid amplification solid-phased reagent are basically the same, and the difference is MgCl 2 It is 3mmol / L and SYBR Green I fluorescent dye is 10umol / L.

Example Embodiment

[0071] Embodiment three:

[0072] A primer, probe sequence and immobilization kit based on PCR detection of Tilletia dwarf (TCK) fluorescent labeled probe, which can be used for the rapid detection of Tilletia dwarf (TCK), and its composition is :

[0073] [1] Sample extraction reagents: 100 mL of TES buffer; 100 mL of 70% ethanol; 10 mL of nucleic acid eluate.

[0074] [2] Nucleic acid amplification solid-phased reagent: It is a solid-phased lyophilized PCR amplification reagent prepared by vacuum freeze-drying using macromolecular stabilizers and can be stored and transported at room temperature. Its components contain the following reagents:

[0075]Component final concentration

[0076] PCR buffer 1X;

[0077] 25mM MgCl 2 2.5mmol / L;

[0078] 25mM dNTP 0.2mmol / L;

[0079] 25uM primer pair 0.5umol / L each;

[0080] Dual-labeled fluorescent probe 0.2umol / L;

[0081] 5Unit / uL Taq polymerase 1Unit / 25uL;

[0082] Biological macromolecule stabilizer added ...

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 discloses a real-time fluorescent PCR fixed-phase agent box and detecting method of wheat short smut, which is characterized by the following: cloning the sequence character segment of relative end particle in the gene group DNA of Tilletia controversa Kuhn TCK according to the RM-PCR; developing the agent box based on specific primer, probe and real-time fluorescent PCR fixed-phase of differential segment sequence; detecting TCK teleutosorus or infective mycelium; producing result from preparing for 4 h.

Description

technical field [0001] The invention relates to an agricultural molecular biology, in particular to real-time fluorescent quantitative PCR detection of specific telomere-related sequences of Tilletia controversa Kühn (TCK), detection primer pairs, probes and solid-phase kits and It is suitable for the rapid detection technology of port inspection and quarantine and early diagnosis of field diseases. Background technique [0002] Tilletia controversa Kühn (Tilletia controversa Kühn, TCK, hereinafter referred to as TCK), belongs to Basidiomycotina, Teliospora, Ustilagoles, Tilletia genus, is a disease of wheat dwarf smut (wheat dwarf bunt) disease) pathogenic bacteria. It is mainly distributed in the Americas, Europe, North Africa and West Asia, especially in the winter wheat regions of the seven states of Montana, Utah, Iowa, Colorado, Washington, and Oregon in the northwestern United States. This disease is the most harmful and extremely diffi...

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
IPC IPC(8): C12N15/31C12Q1/68C07H21/00
Inventor 王中康殷幼平夏玉先袁青曹月青王春林彭国雄曾德玉
Owner CHONGQING UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap