LAMP primer set for detecting jujube witches disease, locust tree witches broom disease and cherry leaching yellowing phytoplasma

A detection kit and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbiological determination/inspection, etc., can solve the problems of restricting the use and promotion of diagnostic methods, complex operating procedures, expensive equipment, etc. Achieve the effect of simple operation, high sensitivity and good stability

Pending Publication Date: 2016-11-30
INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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Problems solved by technology

However, the above-mentioned methods that have been established at present often require expensive instruments and equipment, and the operation procedures are complicated, the de

Method used

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  • LAMP primer set for detecting jujube witches disease, locust tree witches broom disease and cherry leaching yellowing phytoplasma
  • LAMP primer set for detecting jujube witches disease, locust tree witches broom disease and cherry leaching yellowing phytoplasma
  • LAMP primer set for detecting jujube witches disease, locust tree witches broom disease and cherry leaching yellowing phytoplasma

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The preparation of embodiment 1 primer

[0050] 1. Design and synthesis of primers

[0051] The nucleic acid sequences of the tuf genes of different groups of phytoplasma were collected in GeneBank, and DNAMAN software was used to compare and compare the above sequences to find out the phytoplasma subgroups of jujube madness, pagoda tree arbuscular disease and cherry lethal yellowing 16SrV-B Using the LAMP primer online design software PrimerExplorer V4 to design a set of specific primers for the 6 regions of the phytoplasma tuf conserved sequence of the 16SrV-B subgroup, including a pair of Primers, a pair of internal primers and a pair of loop primers, the primers were synthesized by Shanghai Sangon Bioengineering Company according to the HPLC purity level.

[0052] The primer sequences are as follows:

[0053] JWB1-F3:5'-TGATGGTGATAACATACCTATT-3' (SEQ ID No.1);

[0054] JWB1-B3:5'-TTGTGGACGATAATTTATCTCCG-3' (SEQ ID No. 2);

[0055] JWB1-FIP: 5'-CGTCCTGTAGCAACAGTT...

Embodiment 2

[0061] Example 2 Establishment of a rapid detection method of ring-mediated isothermal amplification for jujube mad disease phytoplasma

[0062] 1. Extraction of total plant DNA

[0063] The plant genome extraction kit (Plant Genomic DNA Kit, Adelaide Biotechnology Co., Ltd.) was used to extract the total DNA of the plants to be tested, and stored in a -20°C refrigerator for later use.

[0064] 2. LAMP amplification

[0065] LAMP primers were prepared from Example 1. The loop-mediated isothermal amplification reaction solution is 45 μL per tube, containing: 10 μM JWB1-F3 0.5 μL, 10 μM JWB1-B3 0.5 μL, 40 μM JWB1-FIP 1 μL, 40 μM JWB1-BIP 1 μL, 20 μM JWB1-LoopF ​​1 μL, 20 μM JWB1- LoopB 1μL, LAMP reaction solution RM (2×) 12.5μL, 8U / μL Bst DNA polymerase 1μL, fluorescent dye 10×SYBR GreenⅠ0.5μL, test sample DNA 1μL (DNA concentration between 50-2000ng / μl can be Complete the detection), make up to 25 μL with ultrapure water, and add 20 μL of sealing solution at the same time. ...

Embodiment 3

[0069] Example 3 specificity test

[0070] According to the loop-mediated isothermal amplification reaction system established in the above-mentioned Example 2, the pathogenesis and healthy tissue samples of Paulownia arbuscules, Neem arbuscules, mulberry wilt, periwinkle greening and lettuce yellowing were detected simultaneously in the 16SI group; 16SII The pathogenic and healthy tissue samples of peanut arbuscular, sweet potato arbuscular and stinky cabbage arbuscular in group 16SXIV; the pathogenic and healthy tissue samples of chestnut yellowing in group 16SXIV; and diseased and healthy tissue samples of Arbus chrysogenum. ddH 2 O was used as a negative control for the specificity test of jujube mad disease, arbuscular disease of pagoda tree and cherry lethal etiolation phytoplasma ring-mediated isothermal amplification.

[0071] The pathogenic and healthy tissue samples of Paulownia arbuscular, neem arbuscular, mulberry shrunken disease, periwinkle green change and let...

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Abstract

The present invention discloses a LAMP primer set for detecting jujube witches disease, locust tree witches broom disease and cherry lethal yellowing phytoplasma, and its detection kit and application. The LAMP primer set is designed for detecting jujube witches disease, locust tree wilt disease and cherry lethal yellowing phytoplasma for 16SrV-B group phytoplasma tuf gene conservative sequence. The invention also discloses a LAMP detection kit for jujube witches disease, locus tree witches broom disease and cherry lethal yellowing phytoplasma. The LAMP primer set is used to establish the LAMP detection method for jujube witches disease, locust tree witches broom disease and cherry lethal yellow phytoplasma, and carries out ring-mediated isothermal amplification on the sample DNA to be tested, and determines whether the tested sample is infected with phytoplasma according to the amplification result samples. The detection method established in the invention can be used for detection of jujube witches disease, locust tree witches broom and cherry lethal yellow phytoplasma, and can better distinguish phytoplasma between different groups and inside groups, and has the advantages of good stability, specificity and high sensitivity.

Description

technical field [0001] The present invention relates to the detection of phytoplasma, in particular to a LAMP primer set for detecting jujube madness, pagoda tree arbuscular disease or cherry lethal etiolation phytoplasma, and also relates to the use of the LAMP primer set in detecting jujube madness and pagoda tree arbuscular disease Or the application in phytoplasma lethal etiolation of cherry belongs to the field of LAMP detection of jujube mad disease, arbuscular disease of pagoda tree and phytoplasma lethal etiolation of cherry. Background technique [0002] Phytoplasma (Phytoplasma, formerly known as Mycoplasma-like organism, referred to as MLO), is a class of difficult-to-culture prokaryotes similar to plant pathogenic bacteria but without cell walls, which can cause many important food crops, vegetables, ornamental plants and fruit trees and other economic trees. severe disease. According to the 16S rDNA gene sequence of phytoplasma, phytoplasma can be divided into ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 王圣洁林彩丽王胜坤田国忠于少帅王曦茁汪来发朴春根李永郭民伟
Owner INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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