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Primer, probe and method for qualitatively and quantitatively detecting witches' broom phytoplasma

A quantitative detection, phytoplasma technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of inability to distinguish and identify phytoplasma, and achieve high accuracy and specificity , the effect of high sensitivity

Inactive Publication Date: 2015-04-08
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, a real-time fluorescent PCR detection method for detecting members of the phytoplasma elm yellow group has been established in China. distinguish and identify

Method used

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  • Primer, probe and method for qualitatively and quantitatively detecting witches' broom phytoplasma
  • Primer, probe and method for qualitatively and quantitatively detecting witches' broom phytoplasma
  • Primer, probe and method for qualitatively and quantitatively detecting witches' broom phytoplasma

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Embodiment 1

[0050] The preparation of embodiment 1 primer and probe

[0051] 1. Design and synthesis of primers and probes

[0052] The primers and probes for the real-time fluorescent quantitative PCR detection of jujube mad disease phytoplasma are designed and synthesized as follows: firstly, the 16S rDNA nucleic acid sequences of all phytoplasmas and representative bacteria are collected in NCBI, and the above-mentioned sequences are analyzed by Omiga software. Carry out comparison and consistency analysis to find out the gene difference sites between Jujube phytoplasma and other phytoplasma, use the software Beacon Designer7.0 to screen out a pair of primers, and set a fluorescent Tapman in the amplification region of the primer pair For the probe, the reporter fluorescent dye is labeled at the 5' end of the probe, and the quencher fluorescent dye is labeled at the 3' end of the probe. After the PCR primers and probes specific to the phytoplasma of jujube mad were designed, online ho...

Embodiment 2

[0060] After the primers and probes were synthesized, they were diluted to 10 μmol / L with sterilized double-distilled water, and the primers and probes were stored in a -20°C refrigerator in the dark for future use. Example 2 Establishment of real-time fluorescent PCR detection method for jujube mad disease phytoplasma

[0061] 1. Extraction of total plant DNA: Plant Genomic DNA Kit (TIANGEN) was used to extract total DNA from jujube leaves, and stored in a -40°C refrigerator for later use.

[0062] 2. PCR amplification of 16S rDNA of phytoplasma jujube mad disease

[0063] The phytoplasma 16S rDNA universal primer pair R16mF2: 5'-CATGCAAGTCGAACGGA-3', R16mR2: 5'-CTTAAC CCCAATCATCGA-3' was used for routine PCR amplification of the total DNA of jujube leaves extracted above. The reaction system is 25 μL: 1.0 μL of DNA template, 1.0 μL of each 10 μmol / L primer, 1.0 μL of 10 μmol / L dNTP, 2.5 μL of 10×PCR Buffer, 0.3 μL of 2.5 U / μL Taq DNA polymerase, sterilized ddH 2 O18.2 μL. ...

Embodiment 3

[0082] Example 3 Application of real-time fluorescent PCR detection method for jujube mad disease phytoplasma

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Abstract

The invention belongs to the technical field of plant quarantine, and relates to a primer, a probe and a method for qualitatively and quantitatively detecting witches' broom phytoplasmas. The method comprises the following steps: according to difference between 16S rDNA gene sequences of the witches' broom phytoplasmas and other phytoplasmas, designing a primer pair JWB Primer-F: 5'-TGGTGAGGTAAAGGCTTA-3' / JWB Primer-R: 5'-CTCCCGTAGGAGTTT GG-3' and a TapMan probe JWB-Probe: 5'-FAM-AATGTGGCTGTTCAACCTCTCA-TAMRA-3' for specifically detecting the witches' broom phytoplasmas; measuring a Ct value by an established real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of the witches' broom phytoplasmas; according to a positive decision criteria of a real-time fluorescent quantitative PCR detection result, qualitatively detecting the witches' broom phytoplasmas; calculating the copy concentration of a 16S rDNA gene segment of the witches' broom phytoplasmas in a sample y a fluorescent quantitative PCR standard curve equation established in advance; and obtaining the number of the witches' broom phytoplasmas in the sample so as to achieve quantitative detection. The primer, the probe and the method have the advantages of high sensitivity, strong specificity, good repeatability, high throughput and the like, and can be widely used in plant quarantine, plant protection, scientific research and other fields.

Description

technical field [0001] The invention belongs to the technical field of plant quarantine, and relates to primers, probes and detection methods for the qualitative and quantitative detection of jujube mad disease phytoplasma, in particular to the qualitative and quantitative detection of jujube mad disease phytoplasma by real-time fluorescent quantitative PCR technology. Primers, probes and methods for quantitative detection. Background technique [0002] Jujube witches'broom (JWB), also known as "jujube cancer", is the most serious and devastating disease in jujube production caused by phytoplasma pathogens. Phytoplasmas, formerly known as mycoplasmas, are single-celled prokaryotic organisms without cell walls, surrounded by biofilms, and can cause various diseases. According to the analysis of phytoplasma 16S rDNA gene sequence, phytoplasma can be divided into 28 groups. Among them, the phytoplasma of jujube madness, Phytoplasma elm etiolation and Phytoplasma aureus, which...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/06
CPCC12Q1/6851C12Q2561/113C12Q2561/101C12Q2563/107
Inventor 罗明韩剑徐金红王同仁张祥林
Owner XINJIANG AGRI UNIV
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