A reagent for assisting identification of tobacco ringspot virus and its application

A tobacco ringspot virus and auxiliary identification technology, which is applied in the direction of microorganism-based methods, microorganism measurement/inspection, microorganisms, etc., can solve the problems of ELISA technology such as cumbersome operation steps, prone to false positives, long detection cycle, etc., and achieve broad Application range and prospects, avoiding false positive results, and improving the effect of accuracy

Inactive Publication Date: 2015-08-05
粟智平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] ELISA technical operation steps are cumbersome, the detection cycle is long, the sensitivity is low, and false positives are prone to occur
Compared with ELISA, PCR gel electrophoresis technology has improved in many aspects, but it is easy to cause pollution
There is no research report on the detection of TRSV by semi-nested-real-time fluorescent PCR

Method used

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  • A reagent for assisting identification of tobacco ringspot virus and its application
  • A reagent for assisting identification of tobacco ringspot virus and its application
  • A reagent for assisting identification of tobacco ringspot virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the preparation of reagent

[0038] 1. Design of primers and probes

[0039] According to the conserved region of the coat protein gene (CP) sequence in the TRSV genome in the NCBI nucleic acid database in the United States, two sets of TaqMAN probes and primers were designed respectively. In this study, three primers were designed (two upstream primers TRSVF1, TRSVF2 and one downstream primer TRSVR) and One Taqman probe (TRSVP).

[0040] 2. Composition of reagents

[0041] 1. Composition of Reagent A

[0042] Reagent A consists of TRSVF1, TRSVR and TRSVP (primers and probes were synthesized by Shanghai Sangon).

[0043] TRSVF1 (upstream primer): 5'-CTCCTGATGCACATGTTGGGT-3' (sequence 1 of the sequence listing);

[0044] TRSVR (downstream primer): 5'-GGGCCTGTTTAGACCTTGACC-3' (sequence 3 of the sequence listing);

[0045] TRSVP (probe): 5'(FAM)-AGACGCAACTGTGACGCTCGCATC-3'(TAMARA); (The nucleotide sequence is sequence 4 of the sequence listing, the 5' en...

Embodiment 2

[0053] Embodiment 2, the application of reagent

[0054] Soybean leaves infected with six viruses (TRSV, BPMV, SBMV, PSV, ToRSV, and TSWV) were taken respectively, and healthy soybean leaves were taken as controls, and reagent A and reagent B prepared in Example 1 were used for specificity determination and sensitivity determination respectively. The amplification efficiencies of reagent A and reagent B were compared.

[0055] 1. Determination of specificity of reagents

[0056] 1. Total RNA extraction and quality control

[0057] Total RNA was extracted from leaves infected with each virus (6 species) and healthy leaves (CK1) with a plant total RNA extraction kit. Use the nucleic acid protein analyzer BioPhotometer to measure its OD value, get its concentration and purity value, and use this to control the quality of nucleic acid.

[0058] 2. Reverse transcription to synthesize cDNA

[0059] The total RNA extracted from the 7 kinds of leaves in step 1 was reverse-transcri...

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Abstract

The invention is aimed at providing a reagent assisting to identify a tobacco ringspot virus (TRSV) and an application thereof. The reagent provided by the invention comprises the component of: a specific primer composed of a DNA expressed in a sequence 1 in a sequence table, a DNA expressed in a sequence 2 in the sequence table and a DNA expressed in a sequence 3 in the sequence table. The reagent further comprises a specific probe with a nucleotide sequence as shown in a sequence 4 in a nucleotide sequence table. The tobacco ringspot virus is an important quarantine pest in our country. According to the reagent and the application, a method for detecting the TRSV through semi-nested-RT-Realtime PCR is created through three nested PCR primers and a TaqMan probe. In the method, the nested PCR technology and the real-time fluorescence PCR technology are organically combined; two PCR systems respectively formed by the three primers and the one probe mutually verify to effectively improve the accuracy of the result; and the real-time fluorescence PCR technology is used for effectively improving the detection sensitivity. The method is accurate, sensitive, simple, convenient and quick, and the detection minimum can be up to 4 fg / ul of total plant RNA.

Description

technical field [0001] The invention relates to a reagent for assisting identification of tobacco ringspot virus and its application. Background technique [0002] Tobacco ringspot virus (TRSV), first discovered and reported in Virginia tobacco in the United States in 1927, has been distributed in more than 50 countries in the world. China is now locally distributed and officially controlled. It is an important quarantine object for entry into my country. my country has intercepted the virus from imported goods such as radish seeds imported from the Netherlands, soybeans imported from the United States, and sunflower seeds imported from Serbia. [0003] Tobacco ringspot virus belongs to the family Comoviridae and is a representative species of Nepovirus. The virus particle is a spherical, equiaxed, icosahedron with a diameter of about 28 nm, and the capsomere is composed of 60 coat protein subunits. TRSV has a wide host range and can infect 246 species of plants in 54 fam...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 粟智平封立平许红岩尹伟力耿金培李金庆杨益娥
Owner 粟智平
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