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Kit for auxiliarily detecting tobacco ringspot virus and application thereof

A tobacco ringspot virus and auxiliary detection technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of false positives, detection, difficult initial template copy number, etc., and achieve easy interpretation , high accuracy, avoiding pollution and damage to the human body

Inactive Publication Date: 2012-04-18
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, ELISA, colloidal gold immunochromatography, common PCR, multiplex PCR, immune capture PCR and other methods are commonly used to detect TRSV at home and abroad, but these methods have limitations of false positive results and difficulty in detecting the copy number of the starting template

Method used

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  • Kit for auxiliarily detecting tobacco ringspot virus and application thereof
  • Kit for auxiliarily detecting tobacco ringspot virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the preparation of kit

[0038] The kit consists of specific primer pairs and specific probes.

[0039] Specific primer pairs are as follows:

[0040] Upstream primer (F): 5'-GAGTTTATTGTACATATAGATACCTGGCG-3' (sequence 1 of the sequence listing);

[0041] Downstream primer (R): 5'-CGAAGTCATGAATGTATCCAGG-3' (SEQ ID NO: 2 in the Sequence Listing).

[0042] The specific probe (TaqMan probe) (the nucleotide sequence is sequence 3 in the sequence listing) is as follows:

[0043] 5'-FAM-TGACTCTCAGGTGCATCCTCCCATGTT-TAMRA-3'.

Embodiment 2

[0045] Embodiment 2, the sensitivity and repeatability determination of kit

[0046] 1. Extraction of viral RNA

[0047] Weigh 0.1g of the diseased tobacco leaves (design specific primers, and have been verified by sequencing, it is confirmed that it contains tobacco ringspot virus and does not contain other tobacco viruses), and use the plant virus nucleic acid nano magnetic beads rapid extraction kit to extract viral RNA . The concentration was determined by NanoDrop ND-1000 Spectrophotometer quantitative analyzer.

[0048] To verify the specific primers of tobacco ringspot virus are as follows:

[0049]TRS V-F: 5'-ATGGGTGCTGTGACAGTTGTTC-3'

[0050] TRSV-R: 5'-GGACAAACACGACACTAGGAAAC-3'

[0051] See sequence 4 in the sequence listing for the sequencing results.

[0052] 2. Synthesis of cDNA

[0053] When performing 10-fold concentration gradient dilution on viral RNA, each gradient was repeated 3 times.

[0054] Synthesis system (20.0μL):

[0055] DEPC-H 2 O 7.8 μL ...

Embodiment 3

[0068] Embodiment 3, the specificity determination of kit

[0069] Using the cDNA of four viruses (ArMV, PVX, PVY and TRSV) as templates, real-time fluorescent PCR amplification, the method is the same as in Example 2.

[0070] see results figure 2 . The results showed that only TRSV showed a typical amplification curve, and the Ct value reading was 21.5, which was judged as positive, while the amplification of ArMV, PVX and PVY showed a flat straight line, which was judged as negative. It can be seen that the detection with the kit of the present invention has strong detection specificity.

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Abstract

The invention discloses a kit for auxiliarily detecting tobacco ringspot virus and an application thereof. The kit comprises a specific primer pair and / or a specific probe; the specific primer pair is a primer pair formed by nucleotide sequences shown in a sequence 1 and a sequence 2 in a sequence table; and the nucleotide sequences of the specific probe are shown in a sequence 3 of the sequence table. The kit can be applied to detecting the tobacco ringspot virus, can detect the tobacco ringspot virus in a fast, accurate and quantitative manner, can judge whether a sample contains virus according to whether a fluorescence signal is generated and can judge the content of the virus in the sample according to the size of the generated fluorescence signal value. The kit has the advantages ofstrong specificity, high accuracy, simple and convenient detection process operation (detection can be carried out by only one real-time fluorescence PCR instrument and electrophoresis does not need to be carried out), short time needed by detection (only three hours are needed from the extraction of the tobacco ringspot virus to the occurrence of detection result) and the like, and is suitable for applications of all quarantine inspection stations in agriculture sectors, all port inspection quarantine bureaus, plant virus research institutions and the like.

Description

technical field [0001] The invention relates to a kit for assisting detection of tobacco ringspot virus and its application. Background technique [0002] TRSV (Tobacco ring spot virus) is a member of the genus Nematode-transmitted polyhedronvirus of the family Comoviridae. Virus particles are equiaxed polyhedrons with a diameter of about 28 nm. Under natural conditions, TRSV has a wide host range and can infect 246 species of plants in 54 families. Because of its diverse ways of transmission and serious harm, it has been listed as a quarantine object by many countries in the world. Currently, tobacco ringspot virus is distributed in more than 50 countries including the United States, India, Indonesia, Japan, China, Iran, the United Kingdom, Bulgaria, the Czech Republic, Denmark, France, Germany, Greece, Hungary, Italy, and the Netherlands. Internationally, countries that list tobacco ringspot virus as quarantine targets include: the former Soviet Union, Germany, Australi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 李明福黄新刘梅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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