Real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al.

A technology of stem canker bacteria and real-time fluorescence, which is applied to the measurement/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc. It can solve the problems of low accuracy, difficult identification, and long period of planting observation, etc. Achieve high accuracy and sensitivity, good specificity and stability, fast and simple detection method

Inactive Publication Date: 2013-04-24
新疆出入境检验检疫局检验检疫技术中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a rapid detection kit for sunflower stem canker in order to solve the problems of long period required for planting observation of sunflower stem canker, difficulty in identification, and low accuracy rate in the prior art

Method used

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  • Real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al.
  • Real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al.
  • Real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al.

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Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1 Primer and probe design

[0020] According to the results of sequencing in our laboratory and the reported Phomora ribosomal gene sequence, the primer and probe design software DNAMAN was used to design primers and probes. The primer sequence is:

[0021] PHDf: 5'-CGTTCAAAAGATTCGATGA-3'

[0022] PHDr: 5'-CAGTGGATCTCTGAGTAA-3'

[0023] The probe sequence is: PHDp: 5'-ACTTTCAACAACGGATCTCTTGGTT-3', the 5' end of the probe is labeled with the reporter fluorescent dye FAM, and the 3' end is labeled with the quencher fluorescent dye TAMRA.

Embodiment 2

[0024] Example 2 sunflower sample detection

[0025] 1. Extraction of DNA from sunflower material

[0026] Sunflower material nucleic acid was extracted by kit method (TaKaRa Universal Genomic DNA Extraction Kit Ver3.0 or DNA Extraction Kit for GMO Detection Ver.2.2).

[0027] 2. Real-time fluorescent PCR reaction system

[0028] Using the total DNA of sunflower material as a template, a real-time fluorescent PCR reaction was carried out.

[0029] 10 μL of Real-time PCR Mix, 2 μL of 5 μM primers (PHDf / PHDr), 5 μL of 2 μM / L PHDp, and 2 ng of sample DNA template were added to the 25 μL reaction system.

[0030] 3. Real-time fluorescent PCR reaction conditions

[0031] After putting the sample tube into the Applied Biosystems7300 fluorescent PCR instrument, set the reaction program: the first step is 50°C, 2min; the second step is 95°C, 10min; then enter the third cycle, 95°C, 10s, 60°C, 1min 40 loops.

[0032] The data is collected at the end of each cycle, and the results ...

Embodiment 3

[0033] Embodiment 3 The specificity experiment of sunflower stem canker pathogen real-time fluorescent PCR detection kit

[0034] Using the total DNA of the sunflower stem canker strain as a template, healthy sunflower leaves and other sunflower diseased plants were used as controls, and the changes in fluorescence intensity were detected by real-time fluorescent PCR.

[0035] Experimental results: The total DNA of the sunflower stem canker strain was used as a template, and obvious changes in fluorescence intensity could be observed by real-time fluorescent PCR detection, while the fluorescence intensity of healthy controls and other sunflower diseased plants did not change. See the results in figure 1 .

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Abstract

The invention provides a real-time fluorescent PCR (polymerase chain reaction) detection kit for Phomopsis helianthi M.Muntanola-Cvetkovic et al. Nucleotide sequences of primers adopted by the kit are shown in a sequence table PHDf&PHDr and a nucleotide sequence of probes is shown in a sequence table PHDp. The kit takes the total DNA of samples as a template, utilizes the primers and the probes to carry out real-time fluorescent PCR amplification, collects data after each cycle ends and judges the results according to the amplification curve after reaction ends. The kit has very good specificity and stability, and the detection method is quick and simple and has high accuracy and flexibility, thus providing guarantees for import and export safety of plant materials possibly carrying Phomopsis helianthi M.Muntanola-Cvetkovic et al.

Description

technical field [0001] The invention relates to biological detection technology, in particular to a kit for detecting sunflower stem canker bacteria. Background technique [0002] The harmfulness of sunflower stem canker (Phomopsis helianthi M.Muntanola-Cvetkovic et al.) was first reported in the United States in 1975, and Minnesota lost 60% of its production due to the disease. From 1979 to 1982, the disease occurred in Yugoslavia, and the yield was lost by more than 50%, and the germination rate, 100-grain weight, oil yield and quality of susceptible seeds were reduced. From 1981 to 1986, Hungary, Romania, and France successively reported the occurrence of the disease. In recent years, countries such as Italy, Bulgaria, former Czechoslovakia, the United States, Canada and Argentina have reported large-scale occurrences of the disease. In 1985, the former Soviet Union discovered the disease for the first time on the sunflower hybrid Soldauer in Uzhgorod District, Transcar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 张祥林王翀陈燕刘彬
Owner 新疆出入境检验检疫局检验检疫技术中心
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