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Multiple real-time fluorescence PCR (Polymerase Chain Reaction) detection method and kit of soybean quarantine virus diseases

A real-time fluorescence and virus technology, applied in the direction of fluorescence/phosphorescence, microbial-based methods, biochemical equipment and methods, etc., can solve the problem of affecting detection accuracy and reliability, unfavorable high-throughput detection, and failure to realize multiple viruses at the same time detection and other issues, to achieve the effect of rapid detection, great significance and strong specificity

Inactive Publication Date: 2010-10-13
SHENZHEN AUDAQUE DATA TECH
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AI Technical Summary

Problems solved by technology

[0007] At present, there are multiple detection methods for the above four viruses, but each virus uses a different system, which is not conducive to high-throughput detection.
And in the detection of the same virus, the errors between different detection systems seriously affect the accuracy and reliability of detection.
In addition, the simultaneous detection of multiple viruses has not been realized in the detection of important virus diseases on soybeans at home and abroad.

Method used

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  • Multiple real-time fluorescence PCR (Polymerase Chain Reaction) detection method and kit of soybean quarantine virus diseases
  • Multiple real-time fluorescence PCR (Polymerase Chain Reaction) detection method and kit of soybean quarantine virus diseases
  • Multiple real-time fluorescence PCR (Polymerase Chain Reaction) detection method and kit of soybean quarantine virus diseases

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0025] Example 1 Multiple real-time fluorescent PCR detection and kit for four important virus diseases on soybean

[0026] A primer, oligonucleotide probe and kit for multiplex real-time fluorescent PCR detection for the detection of four important virus diseases on soybeans are used for the specific detection of four important virus diseases on soybeans.

[0027] First, sequence and compare the gene sequences of coat protein (CP) gene sequences of four viruses, namely tobacco ringspot virus, southern bean mosaic virus, tomato ringspot virus and Arabidopsis mosaic virus. The unique base sites of four viruses that are different from other pathogenic bacteria and related species were identified. The primers designed for real-time fluorescent PCR detection of soybean virus diseases have the following nucleic acid sequences:

[0028] TRSV-ZFP

[0029] The designed oligonucleotide probe for real-time fluorescent PCR detection of soybean virus disease has the following n...

Embodiment 2

[0032] Example 2 Using the quadruple real-time fluorescent PCR detection method to detect four kinds of viral RNA extracted from the host

[0033] The primers and oligonucleotide probes used were those in Example 1.

[0034] The total volume of the reverse transcription PCR amplification (RT-PCR) system is 10 μL, and the components are: 5 μL of RT buffer (TOYOBO company), 0.5 μL of 5 μM primer pair, and 0.5 μL of Enzyme mix.

[0035] Reaction conditions: warm bath at 37°C for 15 minutes.

[0036] The four-fold real-time fluorescent PCR detection reaction is shown in Table 2.

[0037] The detection reaction conditions are shown in Table 3.

[0038] Table 2 Quadruple real-time fluorescent PCR detection reaction system (20ul)

[0039]

[0040]

[0041] Table 3 detection reaction conditions

[0042]

[0043] The RT-PCR products of four viruses were used as positive controls, the RT-PCR products of other similar species of viruses were used as negative controls, and a wa...

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Abstract

The invention relates to a simple, quick, specific and sensitive real-time fluorescence PCR (Polymerase Chain Reaction) detection method and a kit thereof for simultaneously detecting one or more soybean virus diseases of tobacco ringspot viruses, southern bean mosaic viruses, tomato ringspot viruses and arabis mosaic viruses. In the real-time PCR detection method, primer sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 and oligonucleotide probes of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 are used. The real-time fluorescence PCR detection method and the kit thereof are suitable for departments of port inspection and quarantine, agricultural production, plant protection, and the like.

Description

technical field [0001] The invention relates to a simple, rapid, specific and sensitive real-time fluorescence method for simultaneously detecting one or more soybean virus diseases among four kinds of tobacco ringspot virus, southern bean mosaic virus, tomato ringspot virus and Arabidopsis mosaic virus PCR detection method and its kit. Suitable for port inspection and quarantine, agricultural production, plant protection and other departments. Background technique [0002] Soybean (Glycine max) is an important oil crop and food crop in my country, and it is also the main source of edible oil and vegetable protein in the world, and occupies an important position in my country's national economy. At present, my country's soybean production ranks fourth in the world, but it still cannot meet the needs of domestic vegetable oil and feed protein processing. As the number of imported soybeans in my country increases year by year, the risk of various diseases that endanger soybe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12N15/11C12R1/94
Inventor 章桂明陈枝楠程颖慧王颖
Owner SHENZHEN AUDAQUE DATA TECH
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