Primer, kit and quantitative detection method for detecting rot valsa mali miyabe et yamada/valsa mali var.dyri

A quantitative detection method and technology for rotting bacteria, applied in the field of fungal molecular detection, can solve the problems of inability to realize biological reaction primers and PCR amplification, and achieve the effects of good primer specificity and avoiding environmental pollution.

Active Publication Date: 2017-07-18
保定百果优农业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of real-time fluorescent quantitative PCR for the quantitative detection of different bacteria needs to solve the problems of PCR primers and reaction conditions suitable for the DNA of each bacteria. Although there are many empirical rules to guide the design of primers, this is not a process of "following the prescription" , but full of uncertainties, the complexity of biological reactions can easily make the designed primers unable to achieve PCR amplification that can achieve the purpose of detection

Method used

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  • Primer, kit and quantitative detection method for detecting rot valsa mali miyabe et yamada/valsa mali var.dyri
  • Primer, kit and quantitative detection method for detecting rot valsa mali miyabe et yamada/valsa mali var.dyri
  • Primer, kit and quantitative detection method for detecting rot valsa mali miyabe et yamada/valsa mali var.dyri

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Primer Design and Screening

[0037] By comparing the DNA ITS region, β-tubulin gene, and EF-1α elongation factor gene sequences of the apple rot pathogen genome, 13 pairs of primers were designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd. Through specific screening, the specific primer pair VE-F and VE-R were obtained, and the specific amplification effect of the primer pair was as follows: figure 1 shown. It can be seen from the figure that the primers VE-F and VE-R can amplify a clear and single band against the pathogen of apple rot, and the product length is 125 bp. After sequencing and comparison, its sequence is highly consistent with the gene sequence of the pathogen of apple rot. No amplified bands were found for Alternaria, Alternaria, Trichoderma, Fusarium laminarum and Penicillium. By carrying out real-time fluorescent quantitative PCR re-screening on primers VE-F and VE-R, the amplification and melting curves of the apple rot pa...

Embodiment 2

[0040] Example 2 Genomic DNA extraction of apple tree rot fungus 41

[0041] Gently scrape off the mycelium of the apple rot pathogen 41 that just covered the plate on the PDA with a blade, freeze and grind with liquid nitrogen, and extract its DNA by CTAB method: take 0.15 g of the surface-sterilized sample and place it in a 2 mL centrifuge tube. Grind to powder in liquid nitrogen, add 700 μL of extract, and then add 700 μL of phenol:chloroform:isoamyl alcohol mixture, the weight ratio of phenol:chloroform:isoamyl alcohol in the mixture is 25:24:1, shake gently and then centrifuge at 12000 rpm for 15 min, take 400 μL supernatant and add it to a new 1.5 mL centrifuge tube, then add chloroform:isoamyl alcohol mixture with a weight ratio of 24:1 and 40 μLCTAB -NaCl, shake gently and then centrifuge at 12000 rpm for 10 min, take 300 μL of supernatant and add it to another 1.5 mL centrifuge tube, add the same amount of frozen isoamyl alcohol, shake gently and then centrifuge at 12...

Embodiment 3

[0042] Example 3 Acquisition of Positive Recombinant Plasmid Standards

[0043] The DNA of strain 41 was amplified by PCR using primers VE-F and VE-R, and the product was subjected to 1% agarose gel electrophoresis, and the specific band of the target fragment was excised using a DNA gel recovery kit. The gel was recovered, purified and connected to the pUC19 vector, and transformed into Escherichia coli Trans 1-T1 competent cells. The positive clones were picked and cultured, extracted with a plasmid mini-extraction kit, and sequenced for identification. The correctly identified recombinant plasmid was used as a positive recombinant plasmid standard.

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Abstract

The invention discloses a primer for detecting valsa mali miyabe et yamada / valsa mali var.dyri. The primer has the following nucleotide sequence: VE-F:5'-AATGAAGTCAGCATCGTTT-3'as a forward primer, and VE-R:5'-GCTTATCAAGGGCTTATCT-3' serving as a reverse primer. The primer has excellent specificity, can be used for specifically amplifying the genome DNA of valsa mali miyabe et yamada / valsa mali var.dyri, and does not have primer dimer generation and non-specific amplification. The invention further establishes a real-time fluorescent PCR quantitative detection method for valsa mali miyabe et yamada / valsa mali var.dyri by utilizing the primers.

Description

technical field [0001] The present invention relates to the field of fungal molecular detection, in particular to a primer for detecting apple tree / pear tree rot fungus, a kit containing the primer, and a quantitative detection method for apple tree / pear tree rot fungus implemented by means of the primer . Background technique [0002] Apple tree rot fungus ( Valsa mali Miyabe et Yamada) can infect multiple parts such as main branches, trunks, and fruits of apple trees, causing apple tree rot. The disease is widespread and serious in all major apple producing areas in my country. Apple rot pathogen has the characteristics of latent infection, indicating that the distribution of the pathogen is far greater than the epidemic area of ​​the disease, and the field plants are generally infected. The pear tree rot fungus mainly harms the main branches and side branches of pear trees, leading to weakening of pear trees and decline in fruit yield and quality. A researcher col...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6851C12Q1/6895C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 王树桐郭永斌曹克强胡同乐王亚南张凤巧
Owner 保定百果优农业科技有限公司
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