Nested PCR (polymerase chain reaction) detection primer and detection method for Uromyces vignae

A technology for detecting primers and single cells, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Effect

Active Publication Date: 2020-04-03
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] An object of the present invention is to provide the nested PCR detection primer of the cowpea monocyst rust to be used for solving the problem that the traditional disease prevalence forecasting technology is difficult to quickly draw accurate forecasting conclusion; Another purpose of the present invention is to provide the cowpea single cell rust Detection method of nested PCR detection primers for bacteria

Method used

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  • Nested PCR (polymerase chain reaction) detection primer and detection method for Uromyces vignae
  • Nested PCR (polymerase chain reaction) detection primer and detection method for Uromyces vignae
  • Nested PCR (polymerase chain reaction) detection primer and detection method for Uromyces vignae

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Effect test

Embodiment 1

[0030] Example 1: Design of specific primers for PCR detection of P. cowpea and verification of specificity of primers

[0031]1. Extraction of Genomic DNA from Monocystis cowpea and other strains

[0032] The CTAB method was used to extract the genomic DNA of Monocystis cowpea and other bacterial strains. The specific method was as follows: the sample to be tested was ground into powder in liquid nitrogen, and 100 μg of the powdered sample to be tested was put into a 2 mL centrifuge tube, and 900 µL 2% CTAB (cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol / L Tris-HCl, pH=8.0; 20 mmol / L EDTA, pH=8.0; 1.4 mol / L NaCl) and 20-30 μL β-mercaptoethanol [Note: CTAB and β-mercaptoethanol need to be mixed at 65 °C], use a shaker to shake and mix, 65 °C water bath for 30 min (shake the centrifuge tube 5-6 times in the middle, (to fully release the DNA into the buffer), take out the tube, cool to room temperature, add 300 μL phenol to mix evenly, let it stand at room temperatu...

Embodiment 2

[0041] Embodiment 2: Sensitivity determination of cowpea monocell rust nested PCR detection

[0042] 1. Routine PCR detection

[0043] Dilute the genomic DNA of Monocystis vignaceae with sterile ultrapure water, and prepare a series of concentrations of 10 times the order of magnitude for future use. Use UV-ITSF / UV-ITSR to perform PCR amplification on genomic DNA with different concentrations, and evaluate the sensitivity of the primers to the detection of the genomic DNA of Monocys vignaceae. The amplification reaction system and reaction procedure are as follows: PCR reaction system 10 µL , including 5 µL of 2×Taq PCR Master Mix (Beijing Tiangen Biochemical Technology Co., Ltd.), 0.5 µL of 10 nmol / L UV-ITSF / UV-ITSR primers, 1.0 µL of DNA template, and make up to 10 µL. The amplification reaction program was: pre-denaturation at 94 °C for 4 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 7...

Embodiment 3

[0049] Example 3: Nested PCR detection of Adzuki bean leaf rust at different times after inoculation

[0050] Obtaining of diseased tissues by artificial inoculation: select Baoqinghong seeds with full grains and uniform size, accelerate germination in a constant temperature incubator at 25 °C, and sow them in flowerpots with a diameter of 15 cm after the seeds germinate, sow 9 seeds in each pot, and sow Afterwards, they were cultured in a greenhouse under the conditions of 16 h light (25 °C) / 8 h dark (20 °C). When the true leaf expansion was 70-80%, the true leaves of adzuki bean were inoculated by referring to the reported method, and Samples were taken at 0 h, 12 h, 24 h, 48 h, 120 h, and 192 h after inoculation. In order to remove the uredia spores remaining on the surface of the leaves during inoculation, the leaves of each sample were washed with tap water on both sides. Rinse again with sterile water for 2-3 times. After rinsing, blot the surface moisture with sterilize...

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Abstract

The invention relates to a nested PCR (polymerase chain reaction) detection primer and a detection method for Uromyces vignae. The nested PCR detection primer for the Uromyces vignae adopts a specificdetection primer for the Uromyces vignae to amplify genome DNA (deoxyribonucleic acid), and sequences an amplification product to obtain sequence information of a UV-ITS (ultraviolet-internal transcribed spacer) amplification product; the sequence information is used as a template to design the nested PCR primer, and the nested PCR primer is named as UV-MX to obtain the nested PCR detection primer for the Uromyces vignae as shown in SEQ ID NO: 1 and SEQ ID NO: 2. The nested PCR detection primer and the detection method for the Uromyces vignae are simple, convenient and rapid to operate; a result can be judged after the genome DNA of a sample to be detected is subjected to extraction, PCR amplification and conventional agarose electrophoresis; a rapid DNA extraction method is adopted in the whole detection process, and pathogenic bacteria do not need to be subjected to isolated culture, so that the detection time is greatly shortened; and the whole detection process can be generally finished within 6 hours.

Description

[0001] 1. Technical field: [0002] The invention relates to the technical field of early warning detection and prevention of crop diseases, in particular to nested PCR detection primers and a detection method for Vigna monocystic rust. [0003] 2. Background technology: [0004] by the living parasitic fungus Monocystic vignacea ( Uromyces vignae ) caused by adzuki bean rust has a long incubation period, and the pathogen uredospores mainly rely on airflow to spread, and can infect multiple times within a growing season, causing widespread prevalence and damage of the disease. At present, the lack of forecasting system for adzuki bean rust makes it easy to miss the best control period in production, resulting in large-scale outbreaks and epidemics of adzuki bean rust. When adzuki bean rust occurs in large numbers, it can cause serious yield and quality losses. Therefore, applying modern molecular biology techniques to early and rapid diagnosis of adzuki bean rust will provide ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6848C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6848C12Q2549/119C12Q2565/125Y02A50/30
Inventor 殷丽华柯希望徐晓丹左豫虎郭博铖麻晓萱
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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