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

A fungal disease, real-time fluorescence technology, applied in the direction of fluorescence/phosphorescence, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of low specificity, poor repeatability, low detection sensitivity, etc., and achieve specificity Strong, meaningful, fast-detecting effects

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

Problems solved by technology

For example, the morphological detection cycle is long and requires rich experience, and some fungi are still controversial or difficult to distinguish species with similar morphological forms; while the existing molecular detection methods are complicated to operate or have low detection sensitivity, or Poor specificity, poor repeatability, etc.
Moreover, there is currently no multiplex detection method capable of simultaneously detecting the four quarantine soybean fungal diseases

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

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example 1

[0024] Example 1 Quadruple Real-time Fluorescent PCR Detection and Kit of Four Important Fungal Diseases on Soybean

[0025] A primer, oligonucleotide probe and kit for the detection of four important quarantine fungal diseases on soybean by quadruple real-time fluorescent PCR are used for the specific detection of four important quarantine fungal diseases on soybean.

[0026] Firstly, the ribosomal transcription region (rDNAITS region) and translation elongation factor 1a fragments of four soybean fungal diseases including Phomopsis soybean, Phomopsis soybean seed rot, southern stem canker and northern stem canker were sequenced respectively. Alignment analysis to find out the unique base positions of each detection target sequence. The primers designed have the following nucleic acid sequences:

[0027] P.SOJAE-1F

[0028] The designed oligonucleotide probe has the following nucleic acid sequence:

[0029] P.SOJAE-1Q

[0030] The quadruple real-time flu...

Embodiment 2

[0031] Example 2 Using the quadruple real-time fluorescent PCR detection method to detect the DNA extracted from the isolated cultures of four quarantine fungal diseases

[0032] The primers and oligonucleotide probes used were those in Example 1.

[0033] The system of its detection reaction is shown in Table 2

[0034] The detection reaction conditions are shown in Table 3.

[0035] Table 2 Quadruple real-time fluorescent PCR detection reaction system (20μl)

[0036]

[0037] Table 3 detection reaction conditions

[0038]

[0039]

[0040] The templates containing the DNA of the four pathogenic bacteria were used as positive controls, and the DNA templates of similar species were used as negative controls, and a water blank control was set. According to the probe labeling information provided in Table 1, set the corresponding fluorescence collection settings for real-time fluorescent PCR.

[0041] The real-time fluorescent PCR detection results are as follows: the...

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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 fungal diseases of soybean phytophthora parasitica, soybean phomopsis seed rot germs, soybean south stem ulcer germs and soybean north stem ulcer germs. The real-time fluorescence PCR detection method and the kit 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 method for simultaneously detecting one or more soybean fungal diseases among four kinds of soybean Phytophthora soybean, Phomopsis soybean seed rot pathogen, soybean southern stem canker and soybean northern stem canker The real-time fluorescent 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 variou...

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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