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Primer group utilizing LAMP technology to detect witloof pseudomonas and kit and method

A technology for detection of Pseudomonas cichorii, which is applied in the directions of recombinant DNA technology, biochemical equipment and methods, and microbial determination/inspection, can solve the problems of long time for amplification and product detection, and achieves improved sensitivity and improved Specific, adaptable effects

Inactive Publication Date: 2016-11-09
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BIOLOG GEⅢMicroStation microbial identification system, the identification process takes 5-7 days, and requires the purchase of expensive equipment and standard databases
The PCR method is the most widely used method. This method is specific, fast, sensitive, and low in cost, but it needs to purchase professional equipment such as a PCR instrument gel imaging system, and the amplification and product detection time is relatively long (about 3 hours).

Method used

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  • Primer group utilizing LAMP technology to detect witloof pseudomonas and kit and method
  • Primer group utilizing LAMP technology to detect witloof pseudomonas and kit and method
  • Primer group utilizing LAMP technology to detect witloof pseudomonas and kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The design of embodiment 1 primer

[0033] According to the specific region of the 16S rRNA gene (Genbank accession number KU923372) of Pseudomonas cichori that infects tomatoes in my country registered in Genbank, the outer primers F3 and B3 and the inner primers FIP and BIP were designed, and the following primer sets were finally determined:

[0034] F3: 5'-CAGCCGCGGTAATACAGAG-3'

[0035] B3: 5'-GCCACTGGTGTTCCTTCC-3'

[0036] FIP: 5'-GCCCGGGGATTTCACATCCAACGGTGCGAGCGTTAATCGG-3'

[0037] BIP: 5'-ACTGCATCCAAAACTGGCGAGCGCATTTCACCGCTACACAGG-3'.

Embodiment 2

[0038] Example 2 Specific amplification of LAMP primers to Pseudomonas cichorium

[0039] 1. Experimental materials

[0040] With Pseudomonas chicory (strain number is Pc-Gd-3, this strain has been disclosed on GenBank, and the accession number of its 16Sr RNA is KU923372), solanacearum (strain number is SSf-4, this strain has been Disclosed on GenBank, the accession number of its 16SrRNA is FJ744124), Pectin bacillus black rot (the strain number is P1, the strain has been published on GenBank, the accession number of its 16SrRNA is KC695819), Pectobacterium carotovorum subsp.brasiliense (strain The serial number is Pcb-zj-1, and the 16Sr RNA sequence of this strain has been registered in the International Gene Bank (http: / / www.ncbi.nlm.nih.gov, accession number KX377593) as material.

[0041]The Pseudomonas cichorium LAMP detection kit includes the following components: the LAMP primer set designed in Example 1, the LAMP reaction solution, the enzyme solution and the nucleic...

Embodiment 3

[0052] Example 3: Sensitivity determination for detection of Pseudomonas cichorium

[0053] 1. Experimental materials

[0054] Pseudomonas chicory as material.

[0055] Pseudomonas chicory LAMP detection kit is the same as in Example 2.

[0056] 2. Experimental method

[0057] (1) Genomic DNA extraction: the method is carried out according to the bacterial genomic DNA extraction kit;

[0058] (2) Take 10 μL of the above-mentioned genomic DNA in a PCR tube as the template mother solution, and dilute the template mother solution by 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 times, take 1 μL from each serial dilution as a template;

[0059] (3) Prepare the loop-mediated isothermal amplification (LAMP) reaction mixture, including 1 μL of each gradient dilution DNA, 12.5 μL of 2×LAMP reaction solution, 3 μL of each primer FIP and BIP with a concentration of 20 μM, and 1 μL each of primers F3 and B3 with a concentration of 10 μM , Bst DN polymerase 1μL, Fluor...

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Abstract

The invention discloses a primer group utilizing an LAMP technology to detect witloof pseudomonas and a kit and a method. The primer group includes a pair of outer-side primers F3 and B3 and a pair of inner-side primers FIP and BJP, and sequences are respectively shown as SEQ IDNO.1-4. The kit includes the primer group and can also include other detection reagents. The method utilizing the LAMP technology to detect the witloof pseudomonas comprises the steps that a genome DNA of a sample to be tested is used as a template, and LAMP amplification reaction is performed by utilizing the primer group or the kit; after the reaction, the color change of an LAMP amplification reaction solution is observed, if the reaction solution is a green troubled solution, the reaction is regarded as positive reaction; if the reaction solution is an orange transparent solution, the reaction is regarded as negative reaction. The primer group has the following advantages that a result is reliable, the specificity is strong, the sensitivity is high, and the primer group is fast and efficient and simple, convenient and easy to operate.

Description

technical field [0001] The invention belongs to the field of plant protection, and in particular relates to a primer set, a kit and a method for detecting Pseudomonas cichorium using LAMP technology. Background technique [0002] Tomato pith necrosis (Tomato pith necrosis) is a new disease that appeared in the tomato production in mainland my country in recent years. Tomato pith necrosis caused by Pseudomonas cichorii infection was first reported in Ireland in 1974, and the disease has been reported in Turkey, Greece, Tanzania and Taiwan. In 2015, tomato pith necrosis caused by Pseudomonas chicory infection was first discovered in Guangzhou City, Guangdong Province. Field symptoms of tomato pith necrosis include the formation of yellow, brown, or black spots or lesions on the stem surface, petioles, and fruit stalks of the plant, followed by symptoms such as water-soaked, discolored, hollow, and soft rot in the stem pith, eventually leading to tomato pith necrosis. The pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 佘小漫何自福汤亚飞蓝国兵
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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