Establishment of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV)

A ring-mediated isothermal, ring-spot virus technology, applied in the field of temperature amplification technology detection, achieves the effects of rapid detection, clear detection results and strong specificity

Inactive Publication Date: 2013-02-13
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Previously, there have been reports on the detection of Streptococcus pneumoniae, West Nile virus, SARS virus, tomato yellow leaf virus, yam mosaic virus, Mycopla...

Method used

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  • Establishment of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV)
  • Establishment of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV)
  • Establishment of reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV)

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Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Establishment of a reverse transcription loop-mediated isothermal amplification detection method for Prunus necrotic ringspot virus

[0040] A reverse transcription loop-mediated isothermal amplification detection method for Prunus necrotic ringspot virus is established, comprising the following steps:

[0041] LAMP-specific primers designed for the detection of Prunus necrotizing ringspot virus

[0042] According to prior art reports and the conserved sequence of the CP gene of PNRSV isolates published in GenBank, a set of specific primers were designed using the LAMP primer design software Primer Explorer V4, including 1 outer primer and 1 pair of inner primers. The primer sequences are as follows (5' -3′):

[0043] F3: AATCATACCCACGCTGGTG

[0044] B3: AATTCGGGGAGGCACATTC

[0045] FIP: TTCGCAGCCCTTTGTTGAGCC-TTGCAAGAAGTGCCATCCG

[0046] BIP: TAGGGTTTCGAGCGGTGTAGGA-TCACGGTCCAAGTGGTCT

[0047] (2) Extraction of plant total RNA

[0048] Take 0.1 g of fres...

Embodiment 2

[0056] Example 2: Optimization of the LAMP detection system

[0057] Experiments and screening were carried out through the three main factors affecting the construction system: BstDNA polymerase concentration, cDNA addition amount, and reaction time, and gradients were set for each influencing factor. See Table 1.

[0058] Table 1 Bst DNA polymerase, cDNA addition amount, reaction time gradient

[0059]

[0060]

[0061] 1. Effect of reaction time length on RT-LAMP amplification results

[0062] Take 10 identical sterilized PCR tubes, 5 of which are set as negative controls, replace the cDNA template with the same amount of DEPC-ddH2O, and the other 5 tubes are used as positive reaction tubes, and all tubes are set up according to the LAMP reaction system established above. Add the corresponding reagents and templates, respectively. After sealing and mixing, take 1 negative control tube and 1 positive reaction tube respectively and amplify for 30 min, 45 min, 60 min,...

Embodiment 3

[0069] Example 3: Specificity of the LAMP detection system

[0070] According to the above-mentioned embodiment-reaction system, healthy plants, PNRSV, CMV, PVX, PVY, BBWV were detected at the same time, and the specificity of the RT-LAMP method was tested. The RT-LAMP amplification reaction was carried out with the RNA extracted from the extracted healthy plants and plant leaves infected with PNRSV, CMV, PVX, PVY, and BBWV as the detection template. Only positive reactions appeared in the PNRSV reaction tubes, and the other reaction tubes were negative. is negative. The results show that the detection system has strong specificity, can specifically detect PNRSV, and has no cross-reaction with other viruses.

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Abstract

The invention discloses establishment of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection method for Prunus necrotic ringspot virus (PNRSV). According to the RT-LAMP detection method for the PNRSV, four specific primers are designed for six regions of a target gene PNRSV, a type of strand displacement deoxyribonucleic acid (DNA) polymerase is utilized at the constant temperature of around 65 DEG C, and efficient amplification of nucleic acids can be achieved in dozens of minutes. The four specific primers are used for identifying the six specific sequence regions of a target sequence, and therefore high specificity of amplification of LAMP is guaranteed. In the process of LAMP, thermal denaturation of template is not needed, temperature is cycled for a long time, the amplification is carried out under isothermal conditions, a waste of time due to temperature change is not caused, and the reaction speed can be increased by 30-50%. Based on whether visible white precipitate of magnesium pyrophosphate exists in a reaction tube, whether the nucleic acids are amplified can be easily judged. The RT-LAMP method for detection of the PNRSV is high in specificity, and the RT-LAMP method for detection of the PNRSV has the advantages of being rapid, high in efficiency, simple in instrument requirement, simple and convenient to operate, low in cost and easy to popularize and use at the grass-roots.

Description

field of invention [0001] The invention belongs to the technical field of biotechnology virus detection, and specifically relates to the technical field of detection of Prunus necrotic ringspot virus (PNRSV) by a loop-mediated isothermal amplification (LAMP) technique. Background technique [0002] At present, various technologies based on PCR reactions are mostly used for molecular detection of viral nucleic acids, such as conventional PCR, nested PCR, real-time fluorescent PCR, etc., which not only require expensive instruments and equipment such as PCR instruments, electrophoresis instruments, and gel imagers, but also operate The procedures are cumbersome and complicated, the time is long, and the technical requirements for the testing personnel are high, which greatly limits the use and promotion as a rapid diagnostic method. [0003] Loop-mediated isothermal amplification (LAMP) is a constant temperature nucleic acid amplification method developed by Notomi et al. in 2...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 罗明殷智婷韩剑周国辉张祥林董代幸
Owner XINJIANG AGRI UNIV
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