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Specific primer, probe and real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting Clavibacter michiganensis subsp. nebraskensis

A real-time fluorescence quantitative and specific technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of low detection efficiency, unstable identification results of field planting phenotype symptoms, and detection methods of separation and culture methods Complicated operation and other issues to achieve high sensitivity and specificity

Pending Publication Date: 2021-10-22
LANZHOU BAIYUAN GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Separation and culture detection method is complicated to operate, takes a long time and has low detection efficiency, so it is not suitable for rapid detection; the identification results of field planting phenotype symptoms are unstable and time-consuming, etc.; ordinary PCR technology has low sensitivity and cannot detect the PCR process in real time

Method used

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  • Specific primer, probe and real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting Clavibacter michiganensis subsp. nebraskensis
  • Specific primer, probe and real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting Clavibacter michiganensis subsp. nebraskensis
  • Specific primer, probe and real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting Clavibacter michiganensis subsp. nebraskensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Design and synthesis of embodiment 1 primers and probes

[0034] According to the present invention, the highly conserved and specific 16S-23S rRNA gene sequence (Accession number: JN613835.1) of Neizhou wilt of maize disclosed by GeneBank, the nucleic acid sequence database of NCBI, is used as the target sequence and designed by software Primer5.0 and Oligo 7 Primers and Probes. The specific comparison results of primers in NCBI Primer-Blast are shown in figure 1 . Select 2 pairs of primers with high specificity and send them to the company for synthesis, and use the dye method for primer screening. Specific probes were synthesized according to the screened primers, and the fluorescent reporter group of the probe sequence was designed: the fluorescent reporter group labeled at the 5' end was FAM, and the fluorescent quencher group labeled at the 3' end was TAMRA.

[0035] figure 1 It is the result figure of NCBI Primer-Blast of the primer of Example 1.

[0036] Th...

Embodiment 2

[0043] The preparation of embodiment 2 standard positive plasmids

[0044] The target fragment in Example 1 was sent to Wuhan Jincare Company to rapidly synthesize standard positive plasmids to prepare standard products.

[0045] (1) Utilize ultra-micro-volume ultraviolet-visible spectrophotometer (ND5000) of Beijing Biotek Biotechnology Co., Ltd. to measure the synthetic plasmid DNA and measure A 260 、A 280 , according to A 260 / A 280 Determine the purity of the plasmid;

[0046] (2) When performing real-time fluorescent quantitative PCR, "copy number" is required as the unit, so the unit is converted into copies / μL. Plasmid concentration (copy number) calculation:

[0047] Plasmid copies / μL=Avogadro's constant×plasmid moles, where Avogadro's constant=6.02×10 23 copies / mol; the molecular weight of the plasmid=(target gene length+vector length) bp×660 (average molecular weight of each pair of bases); that is, plasmid copies / ul=(6.02×10 23 )×(?ng / ul×10 -9 ) / (DNA length×...

Embodiment 3

[0050] The optimization of embodiment 3 real-time fluorescent quantitative PCR reaction conditions and the drafting of standard curve

[0051] (1) Establishment of real-time fluorescent quantitative PCR detection method

[0052] A reaction system was established by diluting the primers and probes synthesized in Example 1, adding deionized water to dilute their concentrations to 10uM working solution for real-time fluorescent quantitative PCR amplification.

[0053] The PCR reaction system is: 2×AceQU+master mix 12.5ul, 10uM upstream primer 0.4ul, 10uM downstream primer 0.4ul, 10uM probe 0.2ul, template DNA 2ul, ddH 2 O 9.5ul, the total reaction volume is 25ul. With the positive plasmid standard substance of embodiment 2 (concentration is 1.0 * 10 8 copies / ul) as a template, ddH 2 O is a blank control. The real-time fluorescent quantitative PCR reaction conditions are:

[0054]

[0055] figure 2 The amplification curve of the system was established for the present inv...

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Abstract

The invention provides a specific primer and a probe for detecting Clavibacter michiganensis subsp. nebraskensis. The specific primer has the following sequence or a complementary chain sequence of the following sequence: the sequence of an upstream primer is 5'-CAGGGAGATCACGGTGCCAA-3'; the sequence of a downstream primer is 5'-AGGACCCAACAGCGTGCAA-3'; and the sequence of the probe is 5'-ACACACCAGACCGCGTTCCCA-3'. The invention also provides a corresponding real-time fluorescent quantitative PCR kit. The specific primer, the probe and the corresponding kit provided by the invention can be used for real-time fluorescent quantitative PCR detection, and the established method has high sensitivity, high specificity and high stability on the Clavibacter michiganensis subsp. nebraskensis.

Description

technical field [0001] The invention relates to specific primers and probes and a real-time fluorescence quantitative PCR detection kit for detecting Neizhou wilt pathogen of maize. Background technique [0002] Goss's Bacterial Wilt and Leaf Blight of Corn is a serious bacterial disease caused by Clavibacter michiganensis subsp. nebraskensis (Cmn), which can lead to Maize yield losses were as high as 50%. The disease is transmitted over long distances through seed transmission, and has spread to many states in the United States and parts of Canada, but has not been found in my country. The sowing area and yield of corn in my country ranks second in the world, and most of the corn planting areas in my country are suitable for the occurrence and spread of Cmn. Once the pathogen is introduced, it will seriously threaten the safety of food production in my country. Therefore, the prevention and control of corn neizhou Wilt disease is of great significance to ensure the sustain...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 车妍李潇玲辛倩李春杜弢徐全乐沈颂东杨立坤车团结
Owner LANZHOU BAIYUAN GENE TECH
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