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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
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  • 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 inconvenience to storage and transportation. The solid-phase kit developed based on biomacromolecule stabilizer combined with layered coating technology is the main development direction for the standardization and industrialization of molecular diagnostic reagents.

Method used

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  • 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

Embodiment 1

[0057] A primer and a solid-phase kit for real-time fluorescent PCR detection based on SYBR Green I fluorescent dye, used for the specific detection of T. dwarf T. dwarf, consists of:

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

[0059] [2] Solid-phase reagents for nucleic acid amplification:

[0060] The real-time fluorescent PCR reaction reagent immobilization mixture is in the form of dry gel, which can be used after adding water. Its components contain 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 added to 23uL; 9umol / L SYBR Green I fluorescent dye.

[0061] Wherein the oligonucleotide primer pair sequence is: 5'-GAAGCTGGTGGAGGTG-3'

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

[0063] [3] Harmless quantitative standard: Wheat TCK recombinant sub-harmless posi...

Embodiment 2

[0066] A primer sequence and a solid-phase kit based on the detection of T. dwarf T. dwarf T. dwarf SYBR Green real-time fluorescent PCR detection, which is used for the specific detection of T. dwarf T. dwarf. It consists of:

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

[0068] Wherein the oligonucleotide primer sequence is: 5'-TTTCGTTGGGCAGTCT-3'

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

[0070] The sample nucleic acid extraction reagent, quantitative standard and detection supplies are the same as in Example 1, and the nucleic acid amplification solid-phase reagent components are basically the same, except that MgCl 2 3mmol / L, SYBR Green I fluorescent dye 10umol / L.

Embodiment 3

[0072] A kind of primer, probe sequence and solid-phase reagent kit based on PCR detection of Tilletia tritici (TCK) fluorescent marker probe, can be used for the rapid detection of T. kkeratinus (TCK), and it consists of :

[0073] [1] Sample extraction reagent: TES buffer 100mL; 70% ethanol 100mL; nucleic acid eluent 10mL.

[0074] [2] Nucleic acid amplification solid-phase reagent: It is a PCR amplification reagent solid-phase freeze-dried product prepared by vacuum freeze-drying using a macromolecular stabilizer and can be stored and transported at room temperature. Its components include 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] Double-labeled fluorescent probe 0.2umol / L;

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

[0082] Add biomacromolecule stabilizer to 23uL;

[0083] The o...

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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

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Application Information

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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
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