Degenerate primer and method of detecting Y potyvirus virus of potato by using degenerate primer

A potato and virus technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems such as the detection of potato virus genus virus by degenerate primers that have not yet been seen, and achieve a rapid detection method. Accuracy, good primer versatility, and low degeneracy

Active Publication Date: 2014-03-19
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no Touch-down PCR detection method using degenerate primers to detect Potavirus genus virus

Method used

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  • Degenerate primer and method of detecting Y potyvirus virus of potato by using degenerate primer
  • Degenerate primer and method of detecting Y potyvirus virus of potato by using degenerate primer
  • Degenerate primer and method of detecting Y potyvirus virus of potato by using degenerate primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0022] 1. Primer design and synthesis

[0023] According to Potatovirus Y acelery virus Y (AVY), belladonna mottle virus (BeMV), ghost needle mosaic virus (BiMV), bean yellow mosaic virus (BYMV), celery mosaic virus (CeMV), pepper ringspot Virus (ChiRSV), Pepper Vein Mottle Virus (ChiVMV), Clover Yellow Vein Virus (CYVV), Johnson Grass Mosaic Virus (JGMV), Konjac Mosaic Virus (KoMV), Maize Dwarf Mosaic Virus (MDMV), Potato Virus A ( PVA), Panax Y virus (PaVY), Potato virus V (PVV), Pepper severe mosaic virus (PepSMV), Peruvian tomato mosaic virus (PTV), Pepper vein mottle virus (PVMV), Potato virus Y (PVY) , Soybean Mosaic Virus (SMV), Sweet Potato Feather Mottle Virus (SPFMV), Turnip Mosaic Virus (TuMV), Tobacco Vein Band Mosaic Virus (TVbMV), NIb, CP Genes of Watertan Mosaic Virus (WTMV) Sequence design primers, the primer sequences are:

[0024] Forward primer: 5'-GTITGYGTKGAYGAYTTYAAYAA-3' (as shown in SEQ ID NO. 1);

[0025] Reverse primer: 5'-GTRTGBCKYTCIGTRTYYTC-3' ...

Embodiment 2

[0038] Versatility detection of the method of the present invention

[0039] Take papaya ringspot virus (PRSV), papaya deformed mosaic virus (PLDMV), pepper ringspot virus (ChiRSV), pepper vein mottle virus (ChiVMV), sweet pepper vein mottle virus (PVMV), tobacco vein mosaic virus Virus (TVbMV), sugarcane mosaic virus (ScMV), sorghum mosaic virus (SrMV), water tomato mosaic virus (WTMV) and jasmine T virus (MVT) 10 kinds of virus leaves, extract respectively according to the method described in embodiment 1 Each leaf total RNA, utilize forward and reverse primer to carry out Touch-down RT-PCR reaction by embodiment 1 method again, after reaction finishes, 1% agarose gel electrophoresis detects amplified product, the result shows (see figure 1 , 1 to 10 in the figure all have electrophoresis bands at about 1700bp positions) DNA fragments of about 1700bp were detected in the 10 kinds of leaves infected with the virus. This proves that the method of the present invention can be...

Embodiment 3

[0041] Specific detection of the method of the present invention

[0042] Get the healthy leaves of bougainvillea, periwinkle, hibiscus, cucumber, capsicum, rice, cotton, papaya, banana, sugarcane, extract each leaf total RNA respectively by the method described in embodiment 1, then carry out Touch- down RT-PCR reaction, 1% agarose gel electrophoresis to detect the amplified product after the reaction, the result shows (see figure 2 , there is no electrophoresis band at the position of about 1700bp from 1 to 10 in the figure) No DNA fragment of about 1700bp was detected in the 10 kinds of leaves. This proves that the method of the present invention does not produce cross-reaction with the host plant of the virus, has good specificity, and meets the detection requirements.

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Abstract

The invention discloses a degenerate primer which consists of a forward primer and a negative primer. The nucleotide sequence of the forward primer is 5'-GTITGYGTKGAYGAYTTYAAYAA-3', and that of the negative primer is 5'-GTRTGBCKYTCIGTRTYYTC-3', wherein Y=C/T, K=G/T, R=A/G, B=G/T/C, and I=hypoxanthine. The invention further discloses a method of detecting Y potyvirus virus of potato by using the degenerate primer. The method comprises the following steps: by taking total RNA (Ribonucleic Acid) of a sample as a template, carrying out Touch-down RT-PCR reaction by means of the primer; after reaction, detecting an amplifying product by gel electrophoresis; judging that the sample contains Y potyvirus virus of potato when an electrophoretic band exists in a position about 1700bp. The primer disclosed by the invention is good in universality and quick and accurate in detection method.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, and in particular relates to a degenerate primer and a method for detecting potyvirus genus virus using the degenerate primer. Background technique [0002] Potatovirus Y ( Potyvirus ) is the family Potatoviridae ( Potyviridae ) is the largest genus. According to the classification standard published by the International Committee on Taxonomy of Viruses in 2012, 146 confirmed species and 32 tentative species have been reported in this genus. Potatovirus Y viruses can pose a serious threat to agricultural production safety. The main transmission route of this virus is the non-persistent transmission of aphids. Generally, the host range is narrow, but most viruses are widely distributed; there are a few other viruses that have a very wide range. host range and can cause significant yield reductions in a variety of crops. According to the "List of Imported Plant Quarantine Pests o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 章绍延王健华刘志昕张雨良余乃通张秀春周朋梁洁
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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