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Special primer capable of detecting different strains of potato viruses and design method thereof

A potato virus and detection method technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of affecting detection results and low specificity of primers, and achieve good repeatability and sample The effect of simple preprocessing and easy operation

Inactive Publication Date: 2011-08-10
SICHUAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The specificity of the primers has a great influence on the reaction. If the specificity of the primers is not high, non-target bands may be generated during the amplification process, which will affect the determination of the detection results.

Method used

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  • Special primer capable of detecting different strains of potato viruses and design method thereof
  • Special primer capable of detecting different strains of potato viruses and design method thereof
  • Special primer capable of detecting different strains of potato viruses and design method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Design of special primers.

[0064] Take PVM as an example:

[0065] 1. Retrieve all reported, discovered and submitted PVM sequences through NCBI's GENBANK database (http: / / www.ncbi.nlm.nih.gov / );

[0066] 2. After downloading as many PVM sequences as possible, use DNAMAN software for comparative analysis to screen out the PVM virus sequences of different strains;

[0067] 3. Compare the PVM virus sequences of different strains by DNAMAN software, and find their conserved regions (that is, all strains are exactly the same, and there is no variation in a DNA sequence);

[0068] 4. Then use PRIMER5, OLIGO and other primer design software to design screening primers in the conserved region;

[0069] 5. Submit the designed alternative primers to NCBI's BLAST system for electronic PCR screening (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome) , select the primers whose similarity is 100% and can search out the most different strains of PVM vi...

Embodiment 2

[0075] Single-plex RT-PCR standardized detection method.

[0076] The RT-PCR detection step is an existing conventional step, and the following are related detection conditions.

[0077] 1. Reverse transcription reaction: In a 20 μl system, 2.5 μl of RNA, 0.5 mM of dNTP, 5 U of RNA inhibitor, 0.5 μl of downstream primer, and 50 U of MMLV reverse transcriptase.

[0078] The RT reaction conditions are: 42°C for 50 minutes, 94°C for 5 minutes, 4°C, and store.

[0079] 2. PCR reaction: in 25μ system, cDNA 4μl, dNTP 0.2mM, Mg 2+ 1.5mM, 0.4μM each for upstream and downstream primers, 1U Taq enzyme.

[0080] The PCR reaction conditions were: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s; annealing at 59°C for 30 s, extension at 72°C for 30 s, a total of 30 cycles; final extension at 72°C for 10 min, and storage at 4°C.

Embodiment 3

[0082] Double RT-PCR standardized detection method.

[0083] The RT-PCR detection step is an existing conventional step, and the following are related detection conditions.

[0084] 1. Reverse transcription reaction: In 20μl system, RNA 2.5μl, 10mM dNTPs 2μl, RNA inhibitor 10U, 5×MMLV buffer 4μl, 20pM viral downstream primers 0.5μl, MMLV reverse transcriptase 100U.

[0085] The RT reaction conditions are: 42°C for 30 minutes, 94°C for 5 minutes, 4°C, and store.

[0086] 2. PCR reaction: in 25μl system, cDNA 4μl, 10mM dNTP 0.5μl, 10×PCR buffer 2.5μl, 25mM Mgcl 2 2 μl, 1 μl each of 20pM virus upstream and downstream primers, 1.5U Taq DNA polymerase.

[0087] The PCR reaction conditions were: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s; annealing at 59°C for 30 s, extension at 72°C for 1 min, 30 cycles; final extension at 72°C for 10 min, storage at 4°C.

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Abstract

The invention discloses a special primer capable of detecting different strains of potato viruses and a design method thereof. The special primer provided by the invention comprises at least one of a primer pair I, a primer pair II, a primer pair III, a primer pair IV, a primer pair V and a primer pair VI in a sequence table. The invention also provides a method and condition for detecting the potato viruses by using the special primer. The invention also provides a design method of the special primer. RC-PCR (Rapid cDNA Cloning- Polymerase Chain Reaction) detection based on the special primer has high specificity, high sensitivity, high detection speed, simplicity in sample pretreatment and high repeatability, is easy and convenient to operate and is easy to analyze; the special primer screened by means of the design provided by the invention has 100 percent of similarity to the potato viruses, and can be used for detecting different strains of potato viruses which are reported or found; and the special primer has a wide application value in the detection of potato viruses.

Description

technical field [0001] The invention belongs to the field of a special primer for virus detection and a design method thereof, and in particular relates to a special primer capable of detecting different strains of potato viruses and a design method thereof. Background technique [0002] Enzyme chain reaction (Polymerase Chain Reaction, PCR) is an in vitro nucleic acid amplification technique developed in the mid-1980s. The PCR reaction is similar to the natural replication process of DNA, consisting of three basic reaction steps of denaturation-annealing-extension, that is, under the action of TaqDNA polymerase, dNTP is used as the reaction raw material, the target sequence is used as the template, and base pairing and semi-retention The principle of replication is to synthesize a new semi-conservative replication strand complementary to the template DNA strand. Through PCR, the target gene or a certain DNA fragment to be studied can be amplified to 100,000 or even a milli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12N15/10
Inventor 宁红孟兴李霄林张洪峰万佳刘可张敏
Owner SICHUAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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