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Odontoglossum ringspot virus RT-LAMP detection primer and detection method thereof

A tooth blue ringspot virus, RT-LAMP technology, applied in biochemical equipment and methods, DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of high experimental equipment requirements, low sensitivity, unfavorable promotion, etc., to achieve Good application prospects, high application value, time-saving effect

Inactive Publication Date: 2017-02-01
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional electron microscopy method is time-consuming and laborious, and requires experienced professionals to identify it. ELISA is currently a more commonly used method, but it also has the defect of low sensitivity. PCR technology with higher sensitivity has become a routine detection method. However, due to the high requirements for experimental equipment, it is not conducive to popularization to grassroots inspection and quarantine departments

Method used

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  • Odontoglossum ringspot virus RT-LAMP detection primer and detection method thereof
  • Odontoglossum ringspot virus RT-LAMP detection primer and detection method thereof
  • Odontoglossum ringspot virus RT-LAMP detection primer and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Dental ringspot virus RT-LAMP detection method, the steps are as follows:

[0038] Step 100: According to the relatively conservative CP gene sequence of ORSV published by GenBank, use the LAMP primer design software primer explorer 4.0 (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design primers for the target gene, and obtain 6 4 specific primers for the region. The 4 specific primer pairs are specifically:

[0039] Outer primer F3: 5'-TGTCTTACACTATTACAGACCCG-3';

[0040] Outer primer B3: 5'-TCATAGCGATAAACTCTGAAGT-3';

[0041] Internal primer FIP: 5'-GGAACTGATTACCCAGAGAATTGGTA-gaattc-TTATTTAAGCTCGGCTTGGGCTG-3';

[0042] Internal primer BIP: 5'-CTGTTCAACAGCAGTTTGCTGATG-gaattc-GCAGGGAACCTACTGGTCAAAG-3'.

[0043] The CP gene sequence of the blue ringspot virus is shown in SEQ ID NO.1; the nucleotide sequence of the outer primer F3 is shown in SEQ ID NO.2, and the nucleotide sequence of the outer primer B3 is shown in Shown in SEQ ID NO.3; the nucleotide sequen...

Embodiment 2

[0053] In order to confirm the correct amplification of the LAMP reaction, EcoRI restriction sites were introduced into FIP and BIP when designing LAMP primers in this experiment. After the LAMP reaction product in step 400 of Example 1 was digested with EcoRI enzyme at 37°C overnight, the digested product was run on the gel, and the electrophoresis results were as follows: figure 2 As shown, each lane is indicated as follows: M: DL2000 DNA marker, 1: restriction product, 2: negative control, 3: LAMP product. figure 2 It indicated that the ladder-like band amplified by LAMP could be successfully digested by EcoRI, and the size of the digested product was similar to the expected size.

[0054] In order to further verify the correctness of the LAMP product, the digested product was gel recovered, connected to PMD18-T to construct a recombinant plasmid containing the target fragment, transformed with JOM109 competent cells, and the transformed bacteria were sent for testing.

...

Embodiment 3

[0058] Specificity verification of RT-LAMP:

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Abstract

The invention provides an odontoglossum ringspot virus RT-LAMP detection primer and a detection method thereof. The method comprises the following steps: designing primers by taking a CP gene sequence of cymbidium mosaic virus as a target gene, so as to obtain four specific primers in accordance with six regions; by virtue of a DNA strand displacement polymerase, conducting RT-LAMP amplification on the target gene with the application of the specific primers under a constant-temperature condition; and finally, conducting 1% agarose gel electrophoresis analysis, DNA fluorescent dye detection, enzyme restriction verification and cloning and sequencing contrasting on a reaction product. The loop-mediated isothermal amplification technique, which is established in accordance with odontoglossum ringspot virus, provided by the invention has the advantages of being rapid and efficient in amplification, good in specificity, simple and convenient to operate, free from the use of special instruments and the like; the technique has a relatively high application value and has a good application prospect in base and field detection.

Description

[0001] 【Technical field】 [0002] The invention relates to a method for detecting plant viruses, in particular to an RT-LAMP detection primer for blue tooth ringspot virus and a detection method thereof. [0003] 【Background technique】 [0004] Odontoglossum ringspot virus (ORSV) is one of the most serious and common viruses that harm orchids. Orchids such as Jianlan and Molan mainly infect ORSV alone. Orchid ringspot virus infects orchids, which will affect the ornamental and utilization value of orchids, and cause symptoms such as mosaic leaves, deformities, petal discoloration and short flower spikes, which will seriously reduce the quality of orchids, thus affecting the development of orchid industry. At present, there is still no effective medicament for the prevention and treatment of blue ringspot virus. Cultivating non-toxic seedlings or targeted isolation and destruction of diseased plants are important measures to prevent and control viral diseases. This requires vir...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 樊荣辉黄敏玲林兵罗远华钟淮钦
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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