Fast detection method for detecting Actinidia chlorotic ringspot-associated virus (AcCRAV) on basis of RT-LAMP-LFD

A technology of RT-LAMP-LFD and detection methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem that advanced equipment is expensive, affects the healthy development of the kiwifruit industry, and affects the development of kiwifruit industry. Yield and quality issues, to achieve the effect of short detection time, reduce detection cost, and reduce loss

Active Publication Date: 2019-10-01
SICHUAN UNIV
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Problems solved by technology

In 2017, the world's kiwifruit production has reached 4.03 million tons, but the infection of diseases will affect the yield and quality of kiwifruit, and even affect the healthy development of the kiwifruit industry
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  • Fast detection method for detecting Actinidia chlorotic ringspot-associated virus (AcCRAV) on basis of RT-LAMP-LFD
  • Fast detection method for detecting Actinidia chlorotic ringspot-associated virus (AcCRAV) on basis of RT-LAMP-LFD
  • Fast detection method for detecting Actinidia chlorotic ringspot-associated virus (AcCRAV) on basis of RT-LAMP-LFD

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[0038]Loop-mediated isothermal amplification detection technology (loop-mediated isothermal amplification, LAMP) is a highly sensitive strand displacement technology developed by Notomi et al. in Japan under isothermal conditions. It is a molecular detection method for rapid nucleic acid amplification. LAMP detection technology designs specific primers for the six regions of the target gene, including internal primers (FIP, BPI) and external primers (F3, B3), under the action of DNA polymerase (Bst DNA polymerase) with strand displacement activity, at constant temperature (About 60-65°C) conditions to carry out continuous displacement amplification reaction on the target gene, and the nucleic acid amplification reaction can be completed after 30-80 minutes of reaction. Compared with conventional PCR, this method does not require expensive specific equipment, and there is no difference in technical indicators such as sensitivity, specificity, and detection range with conventiona...

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Abstract

The invention discloses primers for detecting Actinidia chlorotic ringspot-associated virus (AcCRAV) on the basis of RT-LAMP-LFD and a detection method. The primers include inner primers FIP and BIP and outer primers F3 and B3. The detection method comprises the following steps of S1, extracting RNA of a to-be-detected sample of kiwifruits; S2, reversely transcribing the RNA of the to-be-detectedextracted sample of the kiwifruits into cDNA; S3, taking the cDNA of the to-be-detected sample of the kiwifruits as a template to carry out isothermal amplification; S4, detecting an isothermal amplification subject obtained in step S3 through an LFD test paper strip, and judging whether or not the to-be-detected sample of the kiwifruits contains Actinidia chlorotic ringspot-associated virus. According to the method, the LAMP primers are designed according to the nucleotide sequence of the coat protein of Actinidia chlorotic ringspot-associated virus, so that fast detection of Actinidia chlorotic ringspot-associated virus is realized, whether or not the virus is contained in a kiwifruit plant or a tissue culture seedling can be fast identified, the purposes of detection, monitoring, prewarning and implementation of targeted measures are achieved, and the loss caused by diseases is reduced.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, in particular to a rapid detection method for detecting kiwifruit chlorotic ring spot-related viruses based on RT-LAMP-LFD. Background technique [0002] Kiwi fruit is one of the most important horticultural products in the world. Because its pulp is rich in vitamin C, minerals and dietary fiber, it is favored by the public. In 2017, the world's kiwifruit production has reached 4.03 million tons, but the infection of diseases will affect the yield and quality of kiwifruit, and even affect the healthy development of the kiwifruit industry. Viral disease is a disease with a long incubation period and huge hidden dangers in production. Actinidia chlorotic ringspot-associated virus (ACRaV) is a negative-sense single-stranded RNA virus belonging to the genus Rowan ringspot virus in Europe. Under natural conditions, the virus can infect kiwifruit, and the leaves show symptoms such as ch...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/94
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2565/625
Inventor 席德慧彭期定杨婷憨宏艳宁佳宸
Owner SICHUAN UNIV
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