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Primer groups, kit and method for detection of apple virus diseases

A technology of apple virus and detection method, which is applied in the agricultural field, can solve the problems of low detection efficiency and sensitivity, and achieve the effect of detection of compound infection, good specificity, and optimized reaction procedures

Pending Publication Date: 2020-04-24
徐世彦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, the embodiment of the present invention provides a primer set, kit and detection method for apple virus disease detection, the main purpose is to solve the problem of low detection efficiency and sensitivity

Method used

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  • Primer groups, kit and method for detection of apple virus diseases
  • Primer groups, kit and method for detection of apple virus diseases
  • Primer groups, kit and method for detection of apple virus diseases

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 (detoxification process)

[0046] In the spring of 2017, from the apple resource nursery of Shanxi Provincial Fruit Tree Breeding Center in Tongchuan New District, the popular apple varieties Liga, Fuji Champion, Changfu 2, Honggailu, Honeycrisp, Qinyang, Pink Lady, Liquanshort were selected for production Fu, rootstocks T337, M9, M26, SH38 and SH40; in April to May, take 1cm stem tips from the vigorously growing mother trees, and use 0.1% HgCl 2 Carry out explant disinfection;

[0047] In August 2017, three generations of Liga, Fuji Champion, Changfu 2, Honggailu, Honeycrisp, Liquan Shortfu, Pink Ms. Qin Yang; rootstocks T337, M9, M26, SH38, SH40 apple test-tube seedling tender stems, inoculated 5 per bottle, 20 bottles of each variety, placed in an artificial climate box, 14 hours of light per day, light intensity 2000-3000lx, humidity of 40%, the starting point temperature of heat treatment is 28°C, and then increase by 1°C every day, when it rises to ...

Embodiment 2

[0057] Embodiment 2 (detection process)

[0058] Total RNA extraction and cDNA synthesis: From January 2018, select Honggailu, Qinyang, Yuhua Zaofu, Fuji champion, Changfu 2, Honeycrisp, Liga, Li after 2 to 3 generations of heat treatment. Quanduanfu, Pink Lady apple varieties and T337, M9, M26, SH38, SH40 rootstock test-tube seedlings, each variety is randomly sampled, the leaves or stems of each bottle of test-tube seedlings are taken to extract RNA in a fume hood, and RNAPlant reagent is used to take 0.1g of materials The total RNA of apples was extracted by DNaseI. After purification by DNaseI, the total RNA had good integrity and high quality, and the degradation of RNA was well avoided; Glycogel electrophoresis was used to detect the integrity of the RNA; ≦1 μg of the purified RNA was reverse-transcribed with the RevertAidFisrtStrandcDNASynthesisKit Reverse Transcription Kit, and cDNA was obtained according to the instructions.

[0059] Primer synthesis: The primer sequ...

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Abstract

The invention discloses primer groups, a kit and a method for detection of apple virus diseases, belonging to the technical field of agriculture. The detection method comprises the following steps: extracting total RNAs of a virus-free test-tube plantlet, and carrying out reverse transcription to obtain cDNAs; carrying out PCR amplification on the cDNAs by utilizing primer pairs used for amplifying ASLSV, ASGV, ASPV, ApMV and ASSVd respectively to obtain amplification products; and carrying out gel electrophoresis on the amplification products, and observing electrophoresis bands, wherein detection results are positive if amplified fragments corresponding to the viruses in size in the amplification products are detected, and detection results are negative if no corresponding amplified fragment exists. According to the invention, a RT-PCR method is employed for detecting the apple viruses, a reaction procedure is optimized, and the annealing temperature suitable for each virus is determined, so whether a sample carries the viruses or not can be rapidly and accurately detected, detection accuracy are 100%, and high accuracy and sensitivity are obtained; and the primer groups, the kitand the method can be used for large-scale tissue culture production, and provide a technical support for breeding of virus-free seedlings.

Description

technical field [0001] The invention relates to the field of agricultural technology, in particular to a primer set, a kit and a detection method for detecting apple virus diseases. Background technique [0002] Apple trees are perennial plants, and they are infected and accumulated a variety of viruses during the long-term reproduction process in nature. At present, there are 39 kinds of apple virus diseases reported in the world, including 25 kinds of dominant viruses and 14 kinds of latent viruses. There are six kinds of apple virus diseases identified in my country, namely apple rust virus (ASSV), apple mosaic virus (ApMv), apple green wrinkle fruit virus (AGrCV), apple chlorotic leaf spot virus (ACLSV), and apple chlorosis virus (ACLSV). Stem pox virus (ASPV) and apple stem groove virus (ASGV), the first three are dominant viruses, and the latter three are latent viruses. It is stipulated in the production of apples that as long as these six viruses are removed, it can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/94
CPCC12Q1/701C12Q1/686C12Q2565/125
Inventor 徐世彦梁建军梁彬
Owner 徐世彦
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