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Primer, probe, kit and method for RT-QPCR detection of fusarium sporotrichioides

A technology of RT-QPCR and Fusarium, which is applied in the field of real-time fluorescent quantitative PCR detection, can solve the problems of time-consuming and laborious, inability to accurately reveal the evolution relationship of species, and many influencing factors, and achieve accurate detection, good sensitivity, and high specific effect

Pending Publication Date: 2020-07-31
LANZHOU BAIYUAN GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For many years, people have studied the polymorphism of the bacteria based on experiments such as morphological characteristics, pathogenicity and vegetative fusion groups, but these methods are time-consuming and laborious and cannot accurately reveal the evolutionary relationship of species
[0003] In the prior art, although multiplex PCR is used to detect Fusarium pseudosporum with good specificity and high accuracy, the PCR results need to be analyzed by agarose electrophoresis, and there are many influencing factors

Method used

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  • Primer, probe, kit and method for RT-QPCR detection of fusarium sporotrichioides
  • Primer, probe, kit and method for RT-QPCR detection of fusarium sporotrichioides
  • Primer, probe, kit and method for RT-QPCR detection of fusarium sporotrichioides

Examples

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Embodiment 1

[0059] This example discloses the preparation method of the 18SrRNA gene standard product of the present invention.

[0060] To establish a real-time fluorescent quantitative PCR method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and high specificity of the reaction must be ensured. The 18SrRNA gene widely exists in Fusarium sporogenes and is highly conserved. The present invention adopts the 18SrRNA gene of Fusarium pseudocladoides as the target sequence. This example mainly uses PCR technology to amplify the 18SrRNA gene of Fusarium pseudosporum, and uses gene recombination technology to connect it to the plasmid vector PUC57 to construct the recombinant plasmid PUC57-18SrRNA, and carry out corresponding PCR identification and sequencing identification, and finally After quantification, it is used as a standard for the method to be established, laying the foundation for the next m...

Embodiment 2

[0129] This embodiment discloses that the fluorescent quantitative PCR kit of the present invention comprises the following components:

[0130] Premix 2×Probe Mix (produced by Nanjing Nuoweizan Biotechnology Co., Ltd., the full name is AceQ U + Probe Master Mix).

[0131] An upstream primer with a final concentration of 10 μM and a downstream primer with a final concentration of 10 μM;

[0132] Probe at a final concentration of 10 μM;

[0133] The recombinant plasmid PUC57-18SrRNA gene serial concentration standard product prepared in Example 1 (the serial concentration of the serial concentration standard product is: 3.1 × 10 9 copies / μL, 3.1×10 8 copies / μL, 3.1×10 7 copies / μL, 3.1×10 6 copies / μL, 3.1×10 5 copies / μL, 3.1×10 4 copies / μL, 3.1×10 3 copies / μL, 3.1×10 2 copies / μL, 3.1×10 1 copies / μL);

[0134] Positive control: the concentration is 3.1×10 10 The recombinant plasmid PUC57-18SrRNA prepared in Example 1 of copies / μL;

[0135] wxya 2 O,ddH 2 O was used...

Embodiment 3

[0147] This embodiment discloses that the kit of Embodiment 2 is used to draw the standard curve of the standard.

[0148] With 18SrRNA gene serial concentration standard substance (the serial concentration of serial concentration standard substance is: 3.1×10 9 copies / μL, 3.1×10 8 copies / μL, 3.1×10 7 copies / μL, 3.1×10 6 copies / μL, 3.1×10 5 copies / μL, 3.1×10 4 copies / μL, 3.1×10 3 copies / μL, 3.1×10 2 copies / μL, 3.1×10 1 copies / μL) as a template, use the primers and probes in the kit to carry out fluorescent quantitative PCR amplification, and set positive and negative controls at the same time.

[0149] Positive control: the concentration is 3.1×10 10 The recombinant plasmid PUC57-18SrRNA prepared in Example 1 of copies / μL;

[0150] Negative control: ddH 2 O.

[0151] The PCR reaction system is as follows:

[0152]

[0153]

[0154] Reaction conditions: Contamination digestion at 37°C for 2 minutes, pre-denaturation at 95°C for 10 minutes, 15 seconds at 95°C, 1 ...

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Abstract

The invention discloses a primer, a probe, a kit and a method for RT-QPCR detection of fusarium sporotrichioides and belongs to the technical field of biology. The nucleotide sequences of an upstreamprimer and a downstream primer are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively., and the nucleotide sequence of the probe is shown in SEQ ID NO:3; and the kit comprises the primer and the probe.The fluorescent quantitative PCR detection method comprises steps as follows: extracting total DNA of a to-be-detected sample; preparing a reaction system; diluting a template in a gradient manner toprepare a standard curve sample and a positive control sample; fluorescent quantitative PCR amplification is performed on the to-be-detected sample, the standard curve sample, the positive control sample and a negative control sample by use of the primer and the probe; and a standard curve is drawn, and a result is calculated. The primer, the probe and the kit have high specificity and good sensitivity and can rapidly and accurately detect the fusarium sporotrichioides.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to primers, probes, kits and methods for real-time fluorescent quantitative PCR detection of Cladosporium fusarium. Background technique [0002] Fusarium sporotrichioides is one of the important pathogens of gramineous crops, which can cause head blight in wheat, oats, barley, etc. The fungus mainly damages the spikes of grain crops, causing the damaged spikes to change color and shrink , the grain weight decreases, which in turn leads to a decrease in yield. pose a serious threat to agricultural production. It is the main pathogenic bacteria of plant root rot, and can cause harm to Panax notoginseng, gastrodia elata, heterophylla, cotton, etc. Cladosporium Fusarium can invade the roots of plants to cause root rot or invade the vascular system of plants to cause wilting and death of plants, and can occur during the whole growth process of plants. Although other st...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6851C12Q2531/113C12Q2561/101C12Q2545/113C12Q2545/114
Inventor 朱建宁沈颂东车团结张镭石勇孙宗科高恺李潇玲郑晓玲
Owner LANZHOU BAIYUAN GENE TECH
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