LAMP primer group for detecting phytoplasma as well as kit of LAMP primer group and application of kit

A technique for planting plasmoids and primer sets, applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., which can solve the limitations of the use and promotion of diagnostic methods, high technical requirements for testing personnel, and complex operating procedures, etc. problems, to achieve the effect of eliminating instrument investment, improving detection sensitivity, and high sensitivity

Inactive Publication Date: 2017-02-15
INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these current methods often require expensive instruments and equipment, and the operation procedures are complicated, the detection time is long, and the technical requirements for the detection personnel are relatively high, which greatly limit the use and promotion of this type of diagnostic method

Method used

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  • LAMP primer group for detecting phytoplasma as well as kit of LAMP primer group and application of kit
  • LAMP primer group for detecting phytoplasma as well as kit of LAMP primer group and application of kit
  • LAMP primer group for detecting phytoplasma as well as kit of LAMP primer group and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Establishment of a Rapid Detection Method for Paulownia Arbuscular Disease Phytoplasma Ring-Mediated Isothermal Amplification

[0060] 1. Extraction of total plant DNA

[0061] 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.

[0062] 2. LAMP amplification

[0063] Reaction system: total volume 45 μL, containing: 10 μM PaWB3-F3 0.5 μL, 10 μM PaWB3-B3 0.5 μL, 40 μM PaWB3-FIP 1 μL, 40 μM PaWB3-BIP 1 μL, LAMP reaction liquid RM (2×) 12.5 μL, 8 U / μL Bst 1 μL of DNA polymerase, 0.5 μL of fluorescent dye 10×SYBR Green Ⅰ, 0.5 μL of sample DNA to be tested, made up to 25 μL with ultrapure water, and added 20 μL of sealing solution at the same time.

[0064] Reaction conditions: Mix the configured reaction tubes and centrifuge, place at 63°C for 60 minutes, and inactivate at 80°C for 5 minutes.

[0065] 3. Judgment ...

experiment example 2

[0068] Preparation and preliminary screening of experimental example 2 primers

[0069] 1. Experimental method

[0070] 1.1 Primer design and synthesis

[0071] The nucleic acid sequences of different groups of phytoplasma 16S genes and tuf genes were collected in GeneBank, and DNAMAN software was used to compare and compare the above sequences to find out Paulownia arbuscules, Neem arbuscules, mulberry atrophy, lettuce yellowing and periwinkle The genetic difference sites and highly conserved regions between the green-shifting (16Sr I group) phytoplasma and other groups of phytoplasma, using the LAMP primer online design software Primer Explorer V4 to design 6 regions of the 16S gene and tuf gene conserved sequences of the 16SrI group phytoplasma specific primers. Among them, 6 sets of primer sets (I-VI) were designed for the phytoplasma 16S gene, and two sets of primer sets (⑦ and ⑧) were designed for the tuf gene. The results are shown in Table 1. level synthesis.

[00...

Embodiment 3

[0084] Example 3 specificity test

[0085] 1. Test method

[0086] According to the loop-mediated isothermal amplification reaction system established in the above-mentioned Example 1, the pathogenic and healthy tissue samples of Paulownia arbuscules, neem arbuscules, mulberry wilt, periwinkle greening and lettuce yellowing in the 16SI group were simultaneously tested; 16SII The diseased and healthy tissue samples of peanut arbuscules, sweet potato arbuscules and stinky cabbage arbuscules in group 16SⅤ; the diseased and healthy tissue samples of jujube madness, pagoda tree arbuscules, cherry lethal yellowing and Chongyang wood arbuscules in group 16SⅤ; 16SXIV The incidence of chestnut yellowing and healthy tissue samples; and to sterilize ddH 2 O As a negative control, the specificity test of Paulownia arbuscular, Neem arbuscular, Mulberry wilting disease, periwinkle greening and lettuce yellowing phytoplasma ring-mediated amplification was performed.

[0087] The pathogenic...

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PUM

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Abstract

The invention discloses an LAMP primer group for detecting phytoplasma as well as a kit of the LAMP primer group and an application of the kit. The LAMP primer group, which is strong in specificity and is constituted by nucleotide sequences shown as SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, is finally obtained by designing six groups of primers for six regions of the conserved sequence of a 16S gene of 16SrI group phytoplasma and designing two groups of primers for six regions of the conserved sequence of a tuf gene of the 16SrI group phytoplasma. The invention further discloses the kit of paulownia witch phytoplasma, Chinaberry witch phytoplasma, mulberry dwarf phytoplasma, lettuce yellow phytoplasma and catharanthus roseus green phytoplasma prepared by virtue of the primer group and the invention also establishes a detection method; the detection method has the advantages of being good in stability, strong in specificity, high in sensitivity and the like; and the method is applicable to the detection of the paulownia witch phytoplasma, the Chinaberry witch phytoplasma, the mulberry dwarf phytoplasma, the lettuce yellow phytoplasma and the catharanthus roseus green phytoplasma.

Description

technical field [0001] The invention relates to a LAMP primer set for detecting 16SrI group phytoplasma, in particular to a detection method for using the LAMP primer set to detect Paulownia arbuscules, Neem arbuscules, mulberry atrophy, lettuce yellowing and periwinkle chlorosis phytoplasma, and also relates to LAMP The application of the primer set in the early diagnosis of Paulownia arbuscularis, Neem arbuscularis, mulberry atrophy, lettuce yellowing and periwinkle chlorosis phytoplasma and the monitoring and identification of pathogenic bacteria belongs to the technical field of plant disease detection, identification and control. Background technique [0002] Phytoplasma (Phytoplasma, formerly known as Mycoplasma-like organism referred to as MLO), is a class of prokaryotic organisms similar to plant pathogenic bacteria but without cell walls. It can cause serious diseases of economic trees such as many important food crops, vegetables, ornamental plants and fruit trees....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107
Inventor 林彩丽王圣洁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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