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Reagent for assisting in identifying potato viruses A and application thereof

An auxiliary identification and potato technology, which is applied in the direction of recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome operation steps, prone to false positives, long detection cycle, etc., and reduce cross-contamination , avoid false positive results, and achieve 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 technology has cumbersome operation steps, long detection cycle, low sensitivity, and prone to false positives; PCR gel electrophoresis technology has improved in many aspects compared with ELISA, but it is easy to cause pollution
There is no research report on the detection of PVA by real-time fluorescent PCR

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the preparation of reagent

[0036] 1. Design of primers and probes

[0037] According to the conserved region of the CP gene sequence in the potato virus A genome (NC_004039) in the NCBI nucleic acid database in the United States, two upstream primers (PVA1F and PVA2F), two downstream primers (PVAIR and PVA2R) and two TaqMam probes (PVA1P and PVA2R) were designed respectively. PVA2P).

[0038] 2. Composition of reagents

[0039] 1. Composition of Reagent A

[0040] Reagent A consists of PVA1F, PVA1R and PVA1P (primers and probes were synthesized by Shanghai Sangong).

[0041] PVA1F (upstream primer): 5'-CTCGCAGAGGCGTACATTGA-3' (sequence 1 of the sequence listing);

[0042] PVA1R (downstream primer): 5'-GGTTGCGTTGAAGACCATACC-3' (sequence 2 of the sequence listing);

[0043] PVA1P (probe): 5'(FAM)-ATGAGAAGTCGTGAGAAACCATACATGCCC-3'(TAMARA); (the nucleotide sequence is sequence 3 of the sequence listing, the 5' end is marked with the reporter fluorescent...

Embodiment 2

[0051] Embodiment 2, the application of reagent

[0052] Get respectively the potato leaf that infects four kinds of viruses (PVA, PVV, PVY and CRSV), get healthy potato leaf as contrast, reagent A and reagent B prepared in Example 1 carry out specificity measurement, sensitivity measurement respectively, and reagent A Compared with the amplification efficiency of reagent B.

[0053] 1. Determination of specificity of reagents

[0054] 1. Total RNA extraction and quality control

[0055] Total RNA was extracted from leaves infected with each virus (4 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.

[0056] 2. Reverse transcription to synthesize cDNA

[0057] The total RNA extracted from the five kinds of leaves in step 1 was reverse-transcribed with a reverse transcription kit to o...

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Abstract

The invention aims at providing a reagent for assisting in identifying potato viruses A 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, a sequence 4 and a sequence 5 of a sequence table as well as a probe A shown as in a sequence 3 and a probe B shown as in a sequence 6. The potato viruses A are important quarantine harmful organisms in China. The invention provides two sets of PCR (Polymerase Chain Reaction) primer probe compositions and establishes a method for detecting PVA (Plyvinyl Aetate) by dual primer probes of -RT-Realtime PCR. By adopting a real-time fluorescent PCR technology, the method effectively improves the sensitivity of the detection. Two sets of primer probes with consistent grain efficiency are mutually verified and determined so as to effectively improve the accuracy of the result and achieve stronger operability in actual detection. 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 A virus and its application. Background technique [0002] Potato virus A (PVA), a member of the genus Potyvirus, mainly occurs in Japan, the United States, and European countries. It is locally distributed in my country and control measures are being taken. PVA is an important entry quarantine plant virus in my country. [0003] Potato virus A's natural host is mainly potato, but it can also infect other Solanaceae plants. PVA infection alone causes mosaic or wrinkled leaves, which has a greater impact on potatoes, and can cause more than 40% yield reduction in severe cases (Cheng Ye, Chen Jiong, Chen Jianping, Genome 3'-terminal sequence determination of potato virus A isolates in the suburbs of Hangzhou and Systematic analysis [J]. Zhejiang Agricultural Journal, 2002, 14 (2): 71-75.). If it is co-infected with other viruses such as potato X, Y virus and potato M virus, the ...

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 耿金培赵文军粟智平鲁闽陈洪俊杨益娥朱水芳
Owner INSPECTION & QUARANTINE TECH CENT OF YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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