LAMP rapid detection method for Xinjiang isolate of phytoplasmas of apricot chlorotic leaf roll

A detection method and phytoplasma technology, which are applied in the directions of biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc. The effect of fast, strong specificity, and improved response speed

Pending Publication Date: 2020-03-13
XINJIANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no report on the detection of Xinjiang isolates of P.

Method used

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  • LAMP rapid detection method for Xinjiang isolate of phytoplasmas of apricot chlorotic leaf roll
  • LAMP rapid detection method for Xinjiang isolate of phytoplasmas of apricot chlorotic leaf roll
  • LAMP rapid detection method for Xinjiang isolate of phytoplasmas of apricot chlorotic leaf roll

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Experimental program
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Effect test

Embodiment 1

[0048] The preparation of embodiment 1 primer

[0049] 1. Design and synthesis of primers: First, collect all phytoplasma in Genebank tuf Gene nucleic acid sequence, use Omiga software to compare the above sequences and analyze the consistency, find out the gene difference sites and highly conserved regions between the Xinjiang isolate of Phytoplasma chlorophyllum and other phytoplasma, use the LAMP primer online design software Primer Explorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html) against apricot chlorotic phytoplasma Xinjiang isolate tuf Multiple sets of primers were designed for the 6 regions of the conserved sequence of the gene, and then a set of specific primers were selected according to comprehensive factors such as the conservation of the primer region, the hairpin structure of the primer, the GC content of the dimer, and the Tm value, including 1 External primers ACLR-F3, ACLR-B3 and 1 pair of internal primers ACLR-FIP, ACLR-BIP were synthesized by Sha...

Embodiment 2

[0056] Example 2 Establishment of the LAMP detection method for the Xinjiang isolate of Phytoplasma chlorotica in apricot

[0057] 1. Extraction of total plant DNA

[0058] The plant genome extraction kit (Plant Genomic DNA Kit, TIANGEN) was used to extract apricot leaf veins or bast branches

[0059] Total DNA was stored in a -40°C refrigerator for later use.

[0060] 2. Optimization and establishment of LAMP detection system

[0061] According to the reported basic principles of LAMP, the present invention preliminarily designs and establishes the LAMP reaction system and procedure of the Xinjiang isolate of Phytoplasma chlorotica chlorophyllum. Then optimize the LAMP reaction temperature, amplification time and the main influencing factors on the construction of the reaction system: the concentration ratio of the outer primer to the inner primer (1:1, 1:4, 1:6, 1:8, 1:10, 1 :12, 1:14); Mg 2+ (0~14.0mmol / L, increasing by 0.5 mmol / L); dNTPs (0.2~1.6 mmol / L, increasing by ...

Embodiment 3

[0066] Example 3 specificity test

[0067] The apricot chlorotic phytoplasma Xinjiang isolate LAMP reaction system established according to the above-mentioned embodiment 2, simultaneously with apricot chlorotic phytoplasma (elm yellow group), jujube phytoplasma (elm yellow group), Positive samples of Phytoplasma aureus (Ulmus yellow group), Paulownia arbuscularis (Aster yellow group), positive samples of Phytoplasma aureus (Aster yellow group), and healthy apricot genomes preserved in the laboratory were used as Template for specificity verification.

[0068] Judgment results such as image 3 , Figure 4 As shown, only the DNA of the Xinjiang isolate of Phytoplasma apricot chlorophyllum was positive, and the emerald green was visible to the naked eye, and the agarose gel electrophoresis showed a characteristic trapezoidal band. However, phytoplasma jujuba, phytoplasma aureus, phytoplasma aureus, phytoplasma phytoplasma Paulownia, phytoplasma arbuscularis, phytoplasma arbus...

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Abstract

The invention discloses a LAMP rapid detection method for the Xinjiang isolate of phytoplasmas of apricot chlorotic leaf roll. According to the invention, four specific primers are designed for six regions of the conserved sequence of a tuf gene from the Xinjiang isolate of phytoplasmas of apricot chlorotic leaf roll, and efficient amplification of nucleic acid can be realized within dozens of minutes at an isothermal temperature of about 61 DEG C by using a strand displacement DNA polymerase; the four primers identify six specific sequence regions of the target sequence, so the high specificity of LAMP amplification is ensured; during LAMP, thermal denaturation of a template is not needed, long-time temperature circulation is achieved, amplification is conducted under an isothermal condition, time waste caused by temperature changes is avoided, and a reaction speed can be increased by 30-50%; and after a reaction is finished, the color change of a reaction tube can be directly observed by naked eyes, or a detection result is judged by gel electrophoresis. The LAMP rapid detection method has strong detection specificity on the Xinjiang isolate of the phytoplasmas of apricot chlorotic leaf roll, and has the characteristics of rapidness, high efficiency, simple instrument requirements, easiness and convenience in operation, low cost and easiness in popularization and applicationin a basic level.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to the technical field of detecting phytoplasma chlorophyllus by using loop-mediated isothermal amplification (LAMP) technology. Background technique [0002] Apricot chlorotic leaf roll (ACLR) is a highly fatal phytoplasmic disease that harms apricot trees. China, Europe and North America have included it in the list of imported plant quarantine pests. The disease was first discovered on French apricot trees in 1973, and was subsequently found on other European stone fruit tree species such as almonds, European plums, cherries, peaches, and nectarines, and became a disease affecting the commercial cultivation of European stone fruit trees. main disease. Although the symptoms of apricot chlorotic leaf roll disease reported in different countries are similar, the pathogens of apricot chlorotic leaf roll disease reported in various countries include a variety of p...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/6806C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844C12Q1/6806C12Q2531/119C12Q2565/125
Inventor 韩剑罗明陈杭张祥林
Owner XINJIANG AGRI UNIV
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