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Method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction)

A potato virus and real-time fluorescence technology, which is applied in the fields of fluorescence/phosphorescence, biochemical equipment and methods, and microbial measurement/inspection, etc. It can solve the problems affecting the specificity of the experiment, the increase of primer design and amplification conditions, and the difficulty of distinguishing melting peaks, etc. problem, to achieve the effect of overcoming the detection sensitivity is not high

Inactive Publication Date: 2013-11-06
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition to the target fragment, the dye can also combine with the primer dimer to affect the specificity of the experiment, which increases the difficulty of primer design, and the commonly used Sybr Green I has the defect of "dye redistribution", which inhibits PCR amplification and has a negative effect on PCR amplification. There is preference in the amplification products. In the melting curve analysis after PCR amplification, it is often very difficult to distinguish the melting peaks of multiple amplification products.
Because of the above-mentioned limitations, every time a virus is added in a detection, the difficulty of primer design and amplification conditions will increase by a level. cause of virus

Method used

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  • Method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction)
  • Method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction)
  • Method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The establishment of embodiment 1, 5 kinds of potato virus basic detection methods

[0068] Step 1: Primer Synthesis

[0069] The 5 kinds of viral primer sequences (see Table 1) designed by the present invention were entrusted to China Shanghai Sangon Biological Company Technology Service Company to synthesize primers, and the synthesis amount was 2OD per primer.

[0070] Step 2: Small amount of total RNA extraction from positive samples

[0071] Put 500mg of each positive potato dry powder sample containing potato PVX, PVY, PLRV, PVA and PVS virus sources into a 5ml centrifuge tube, then add Trizo at a ratio of 1ml Trizol / 100mg, and place the homogenate on ice for 5min. Add chloroform at a ratio of 200ul chloroform / ml Trizol. Shake vigorously for 30s, put on ice for 5-10min, after layering, centrifuge at 12000r / min for 20min at 4°C, take the supernatant into another 5ml centrifuge tube, add an equal volume of isopropanol (about 0.5ml) to lightly Mix gently, place at...

Embodiment 2

[0081] Potato virus detection in the potato leaves of embodiment 2, Hangzhou Gaosha area

[0082] Step 1: Primer Synthesis

[0083] Same as the first step in Example 1.

[0084] Step 2: Leaf Sampling

[0085] The sampling location is the Gaosha area of ​​Hangzhou, Zhejiang. Select the plants with virus symptoms in the potato planting area, and cut the leaves with scissors. Every time a plant sample is taken, the scissors are disinfected with 75% (volume concentration) alcohol, and then the next sampling is performed. Each sample was packed into a polyethylene food bag, and then put into a sampling box for storage and brought back to the laboratory. A total of 20 samples were set.

[0086] Step 3: small amount of total RNA extraction from leaf samples

[0087] The operation steps are equal to the second step of Example 1.

[0088] Step 4: Reverse transcription to synthesize cDNA

[0089] Same as the third step of Example 1.

[0090] Step 5: Multiplex fluorescent quantit...

Embodiment 3

[0095] Embodiment 3, the detection of potato virus in potato tuber

[0096] Step 1: Primer Synthesis

[0097] Same as the first step in Example 1.

[0098] Step Two: Tuber Sampling

[0099] The sampling location for the detection of potato tuber virus was carried out in Haining, Zhejiang Province. A total of 5 varieties were detected in this experiment. Five plants were randomly selected from each variety, and one tuber was taken from each plant. After the tubers were brought back to the laboratory, the soil was washed away with tap water, then washed three times with double distilled water, and dried at room temperature. Cut 5 tubers of each variety into small pieces and mix them with a knife, then take 500 mg for extraction of total RNA. A total of 20 samples were set.

[0100] Step 3: Mini-extraction of total RNA from potato tuber samples

[0101] Take 500 mg of the above potato tuber sample and grind it into powder in liquid nitrogen, then put it into a 5 ml centrifu...

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Abstract

The invention discloses a method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction). The method comprises the following steps: 1), synthesizing specific primers corresponding to a potato virus X--PVX, a potato virus Y--PVY, a potato leaf roll virus--PLRV, a potato virus A--PVA and a potato virus S--PVS respectively; 2), extracting the total RNA of a potato sample, and conducting reverse transcription on the total RNA to synthesize a cDNA; and 3), placing all the specific primers in a polyfunctional fluorescent quantitation (PCR) PCR reaction system, carrying out PCR amplification by taking the cDNA obtained in the step 2 as a template, and analyzing whether the potato sample carries at least one of the PVX, the PVY, the PLRV, the PVA and the PVS or not.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, and in particular relates to a method for simultaneously detecting five kinds of potato viruses by multiplex real-time fluorescent PCR. Background technique [0002] Potato is an important worldwide food crop. Potato virus disease is a very important kind of disease on potato. It is distributed in every potato growing area in the world. Some potato viruses can seriously affect the yield and quality of potato. In my country, the main types of potato virus are potato virus X (Potato virus X, PVX), potato virus Y (Potato virus Y, PVY), potato leafroll virus (Potato virus, PLRV), and potato virus A (Potato virus, PVA). ) and potato S virus (Potato virus, PVS). The viruses that have the greatest impact on production are PVY and PLRV, followed by PVX and PVA, and PVS has less impact. These viruses seriously affect the yield and quality of potatoes, generally reducing yield by 10% to 30%,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 姜永厚程菊会董沁芳郭江峰丁先锋
Owner ZHEJIANG SCI-TECH UNIV
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