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Reagent for assistant identification of carnation ringspot viruses and application thereof

A ringspot virus and auxiliary identification technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of cumbersome operation steps, prone to false positives, and long detection cycle, so as to reduce crossover Contamination, avoidance of false positive results, consistent amplification efficiency

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

ELISA technical operation steps are cumbersome, the detection cycle is long, the sensitivity is low, and false positives are prone to occur
There is no research report on the detection of CRSV by real-time fluorescent PCR

Method used

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  • Reagent for assistant identification of carnation ringspot viruses and application thereof
  • Reagent for assistant identification of carnation ringspot viruses and application thereof
  • Reagent for assistant identification of carnation ringspot viruses and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the preparation of reagent

[0037] 1. Design of primers and probes

[0038] Two upstream primers (CRSV1F and CRSV2F), two downstream primers (CRSV1R and CRSV2R) and Two TaqMan probes (CRSV1P and CRSV2P).

[0039] 2. Composition of reagents

[0040] 1. Composition of Reagent A

[0041] Reagent A consists of CRSV1F, CRSV1R and CRSV1P (primers and probes were synthesized by Shanghai Sangong).

[0042] CRSV1F (upstream primer): 5'-GGTGACAGTGTTGCACTGAACTTT-3' (sequence 1 of the sequence listing);

[0043] CRSV1R (downstream primer): 5'-CCAGGAGTGCCGACAGAAAC-3' (sequence 2 of the sequence listing);

[0044] CRSV1P (probe): 5'(FAM)-TGGCACCCAACGAAAGAACTAAGTCCG-3'(TAMARA); (the nucleotide sequence is the sequence 3 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 ).

[0045] The target sequence size of CRSV1F and CRSV1R is 74bp (see sequence 7 of the sequence...

Embodiment 2

[0052] Embodiment 2, the application of reagent

[0053] Take carnation leaves infected with five kinds of viruses (CRSV, PVA, PVV, PVY and PPV) respectively, and get healthy carnation leaves as a contrast, and reagent A and reagent B prepared in Example 1 are used for specificity determination and sensitivity determination respectively. The amplification efficiencies of reagent A and reagent B were compared.

[0054] 1. Determination of specificity of reagents

[0055] 1. Total RNA extraction and quality control

[0056] 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.

[0057] 2. Reverse transcription to synthesize cDNA

[0058] The total RNA extracted from the six kinds of leaves in step 1 was reverse-transcribed wit...

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Abstract

The invention provides a reagent for assistant identification of carnation ringspot viruses and an application thereof. The reagent comprises a specific primer formed from DNAs shown in a sequence 1, a sequence 2, a sequence 4 and a sequence 5 in a sequence table, a probe A shown in a sequence 3 and a probe B shown in a sequence 6. The carnation ringspot viruses are major quarantine harmful organisms in China. The invention provides two PCR (Polymerase Chain Reaction) combinations of primers and probes, and creates a method for using double real-time PCR combinations of primers and probes to detect the carnation ringspot viruses. The method adopts the real-time fluorescence PCR technology, and effectively increases the sensitivity of detection. The two sets of combinations of primers and probes, which have consistent amplification efficiency, are authenticated by each other, thus, the accuracy of results is increased, and the method has strong operability in actual detection. The method is accurate, sensitive, simple, convenient and quick, and has the low detection limit reaching 0.46 fg / mul of total RANs of plants.

Description

technical field [0001] The invention relates to a reagent for assisting the identification of carnation ringspot virus and its application. Background technique [0002] Carnation ringspot virus (CRSV), belonging to the genus Carnation ringspot virus in the family Tomatobush Viridae, is an important quarantine plant virus ("List of Quarantine Pests for Imported Plants of the People's Republic of China" (2007)). The virus mainly occurs in Finland, Germany, Lithuania, Poland, the United Kingdom, the former Soviet Union, Italy, Hungary, France, the Netherlands, Colombia, Mexico, the United States, Australia and other countries, and has not been reported in my country. [0003] The natural host of carnation ringspot virus is mainly carnation, as well as apple, Prunus, pear, chickweed, bluegrass, etc., which are widely planted in my country. After CRSV infection, the carnation calyx splits, the number of flowers per plant decreases, and the yield is reduced by 25%. After flower...

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 粟智平耿金培杨益娥李晓玉朱水芳王真陈洪俊
Owner INSPECTION & QUARANTINE TECH CENT OF YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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