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Cymbidium mosaic virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection primers and detection method thereof

A technology of RT-LAMP, building orchid leaves, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc. Application prospects, high application value, rapid and efficient amplification

Inactive Publication Date: 2016-12-07
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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  • Cymbidium mosaic virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection primers and detection method thereof
  • Cymbidium mosaic virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection primers and detection method thereof
  • Cymbidium mosaic virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection primers and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] To build an orchid mosaic virus RT-LAMP detection method, the steps are as follows:

[0033] Step 100: According to the relatively conservative CP gene sequence of CyMV 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 targets 4 specific primers for the region. The 4 specific primer pairs are specifically:

[0034] Outer primer F3: 5'-TGGTTTCCAGGAGGATACCC-3';

[0035] Outer primer B3: 5'-AAGATGGCCCTTGGTGACCT-3';

[0036] Internal primer FIP: 5'-ATTCAGCAGGCTCCAGCGCGGCA-gaattc-CCGCCTTTGACTTCTTCGATGC-3';

[0037] Internal primer BIP: 5'-AAGTACGGCGCCCTTGCCCGT-gaattc-GTGATGAGGTTGCCGTTTTGGA-3'.

[0038] The CP gene sequence of the orchid mosaic 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 SEQ ID NO.2 ID NO.3; the nucleotide s...

Embodiment 2

[0048] 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.

[0049] 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

[0053] Specificity verification of RT-LAMP:

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Abstract

The invention provides Cymbidium mosaic virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection primers and a detection method thereof. The primers are as follows: the nucleotide sequence of the outer primer F3 is disclosed as SEQ ID NO.2, the nucleotide sequence of the outer primer B3 is disclosed as SEQ ID NO.3, the nucleotide sequence of the inner primer FIP is disclosed as SEQ ID NO.4, and the nucleotide sequence of the inner primer BIP is disclosed as SEQ ID NO.5. The method comprises the following steps: designing primers by using CP gene sequence of Cymbidium mosaic virus as the target gene to obtain 4 specific primers for 6 regions, replacing the polymerase with a DNA chain, carrying out RT-LAMP on the target gene under homothermal conditions by using the specific primers, and finally, carrying out 1% agarose gel electrophoresis analysis, DNA fluorescent dye detection, enzyme digestion verification and clone sequencing comparison on the reaction product. The LAMP technique established according to the Cymbidium mosaic virus has the advantages of high amplification speed and efficiency, favorable specificity, no need of special apparatuses and the like, is simple to operate, has higher application value, and thus, has favorable application prospects in base layer and field detection.

Description

【Technical field】 [0001] The invention relates to a plant virus detection method, in particular to a Jianlan mosaic virus RT-LAMP detection primer and a detection method thereof. 【Background technique】 [0002] Cymbidium mosaic virus (CyMV) is one of the most serious and common viruses that harm orchids. Cymbidium mosaic virus (Cymbidium, Dendrobium, Oncidium, etc.) mainly infects Cymv alone. Orchid mosaic virus infects orchids, which will affect the ornamental and utilization value of orchids, causing symptoms such as mosaic leaves, deformities, petal discoloration, and short flower spikes, resulting in a serious decline in quality, thereby affecting the development of the orchid industry. At present, there is still a lack of effective agents for the prevention and treatment of Jianlan mosaic virus. Cultivation of non-toxic seedlings or targeted isolation and destruction of diseased plants are important measures for the prevention and treatment of viral diseases. This requ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 樊荣辉黄敏玲钟淮钦叶秀仙
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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