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Reagent for assisting in identifying potato viruses V and application thereof

An auxiliary identification and potato technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of cumbersome ELISA operation steps, expensive electron microscopy equipment, and prone to false positives, so as to avoid false positives. Positive results, broad application scope and prospects, the effect of reducing false positive results

Inactive Publication Date: 2011-04-06
INSPECTION & QUARANTINE TECH CENT OF YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Electron microscopy equipment is expensive and has low detection sensitivity; ELISA technology has cumbersome operation steps, long detection cycle, low sensitivity, and prone to false positives
There is no research report on the detection of PVV by real-time fluorescent PCR

Method used

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  • Reagent for assisting in identifying potato viruses V and application thereof
  • Reagent for assisting in identifying potato viruses V and application thereof
  • Reagent for assisting in identifying potato viruses V and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of reagent

[0035] 1. Design of primers and probes

[0036] One upstream primer (PVVF), two downstream primers (PVVR1 and PVVR2) and one Taqmam probe (PVVP) were designed according to the conserved region of the CP gene sequence in the potato V virus genome (NC_004010) in the NCBI nucleic acid database.

[0037] 2. Composition of reagents

[0038] 1. Composition of Reagent A

[0039] The kit consists of PVVF, PVVR1 and PVVP (primers and probes were synthesized by Shanghai Sangong).

[0040] PVVF (upstream primer): 5'-CCGCAGCTGGTGAGCAAT-3' (sequence 1 of the sequence listing);

[0041] PVVR1 (downstream primer): 5'-GTGCCAGTTGTTCCTGCATTT-3' (sequence 2 of the sequence listing);

[0042] PVVP (probe): 5'(FAM)-CATCCCGTTCTTCGAGACCCTACTT-3'(TAMARA); (the nucleotide sequence is sequence 4 of the sequence listing, the 5' end is marked with the reporter fluorescent dye FAM, and the 3' end is marked with the quencher fluorescent dye TAMRA ).

...

Embodiment 2

[0050] Embodiment 2, the application of reagent

[0051] Get the potato that infects five kinds of viruses (PPV, PVA, PVV, PVY and CRSV) respectively, get healthy potato leaf as contrast, application embodiment 1 prepares reagent A and reagent B to carry out specificity measurement, sensitivity measurement respectively, and carry out reagent Comparison of amplification efficiencies of reagent A and reagent B.

[0052] 1. Determination of specificity of reagents

[0053] 1. Total RNA extraction and quality control

[0054] Total RNA was extracted from leaves infected with each virus (5 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.

[0055] 2. Reverse transcription to synthesize cDNA

[0056] The total RNA extracted from the six kinds of leaves in step 1 was reverse-transcribed wit...

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Abstract

The invention aims at providing a reagent for assisting in identifying potato viruses V and application thereof. The reagent provided by the invention comprises a specific primer consisting of DNAs shown as in a sequence 1, a sequence 2 and a sequence 3 of a sequence table as well as a probe of the nucleotide sequence shown as in a sequence 4. The potato viruses V are important quarantine harmful organisms in China. The invention establishes a method for detecting PVA (Plyvinyl Aetate) by heminested-RT-Realtime PCR(Polymerase Chain Reaction) by using three PCR primers and one TaqMan probe. The method is organically combined with the nested PCR and the real-time fluorescent PCR technology; two sets of PCR systems formed by three primers are mutually verified to effectively improve the accuracy of the result; and a fluorescent probe effectively improves the sensitivity of the detection. Proved by experimental results, the method is accurate, sensitive, simple, convenient and quick; and the detected low limits of the potato viruses can respectively reach 0.5fg / mul of total RNA (Ribonucleic Acid) of plant.

Description

technical field [0001] The invention relates to a reagent for assisting identification of potato V virus and its application. Background technique [0002] Potato virus V (PVV) is an important potato virus that mainly occurs in France, Ireland, Peru, and the United Kingdom. PVV has not been reported in my country, and it is an important quarantine object of my country's entry ("List of Plant Quarantine Pests of Imported People's Republic of China" (2007 edition)). The natural host of potato V virus is potato. It is a very important virus on potato. It can be transmitted non-persistently by a variety of aphids and is very harmful to potato. The virus can also be mixed with other viruses, which is more harmful and can cause a decrease in yield and affect potato production. PVV is systematically infected on potatoes, and the virus can be transmitted to potato cubes. The potato cubes have a high rate of virus infection, and the probability of carrying the virus in the introduc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 粟智平张永江王真李彬李明福杨益娥陈洪俊
Owner INSPECTION & QUARANTINE TECH CENT OF YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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