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Primers for visually detecting ascochyta citrullina of different genetypes and application thereof

A technology for the genotyping of spp., which is applied in the field of primers for the visual detection of different genotypes of spp., can solve the problems of high requirements on instrument conditions, complicated detection process, and long detection time, and achieve low cost, high sensitivity, and easy instrumentation. simple effect

Inactive Publication Date: 2016-03-30
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved: the present invention is aimed at the long detection time required for the biological detection method of Blight blight in the prior art, the fluorescent quantitative PCR technology has high requirements for the instrument conditions, the detection process is complicated, and the cost is high

Method used

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  • Primers for visually detecting ascochyta citrullina of different genetypes and application thereof
  • Primers for visually detecting ascochyta citrullina of different genetypes and application thereof
  • Primers for visually detecting ascochyta citrullina of different genetypes and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] A broad-spectrum, visualized, detection kit for detecting different (RGI and RGII) genotypes of Phytophthora spp.: 40 μM forward inner primer DB17RG-FIP, 40 μM reverse inner primer DB17RG-BIP, 5 μM forward outer primer DB17RG- F3, 5 μM reverse outer primer DB17RG-B3, 20 μM loop primer DB17RG-LB, 10 mM dNTPs, 20 mM Tris-HCl (pH8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 8mMMgSO 4 , 0.1% TritonX-100, BstDNApolymerase8 unit, 8μM Calcein (Calcein), 0.3mMMnCl 2 ; target DNA, add ultrapure water to prepare 25 μL detection solution. Wherein, each primer sequence is specifically as follows:

[0030] DB17RG-FIP: 5'-GTGAGGGCCCTGAGATGTTTG-AATTATTCGCCTACAAGCCGC-3';

[0031] DB17RG-BIP: 5'-CCGCATCCGACATCACCCTT-GCTTCGCCTTCCTCATCG-3';

[0032] DB17RG-F3: 5'-AGACCGCACTTTCGAGCT-3';

[0033] DB17RG-B3: 5'-GCGAACTGGCCAATGTGT-3';

[0034] DB17RG-LB: 5'-TCCACAAGGTCCCGCAAT-3'.

[0035] Forward inner primer DB17RG-FIP, reverse inner primer DB17RG-BIP, forward outer primer DB17RG-F3, reve...

Embodiment 2

[0037] Amplified assay specificity assay for detection of different (RGI and RGII) genotypes of P. solani.

[0038] In order to verify the specific primer sequences of different (RGI and RGII) genotype blight blight bacteria, the present invention uses 3 RGI genotype blight blight bacterial strains and 2 RGII genotype blight blight bacterial strains and 8 kinds of pathogenic fungi as test materials (Table 1), take 2 μL of the DNA solution of the above-mentioned tested strain, add 23 μL of the detection solution prepared in Example 1 to carry out the amplification reaction, and the reaction program is 63° C., 40 min. The results show that based on the color reaction of the reaction system as the result judgment standard, the reaction tubes of the 3 strains of RGI genotype R. blight strains and the 2 RGII genotype strains of R. blight all showed a color reaction of yellow-green ( figure 1 Middle a) and white magnesium pyrophosphate precipitate turbidity ( figure 1 In b), or aga...

Embodiment 3

[0043] Sensitivity test for detection of amplification reactions of different (RGI and RGII) genotypes of P. solani.

[0044] In order to determine the sensitivity of the detection method, the DNA of the extracted Phytophthora spp. was measured with a spectrophotometer, then diluted 10 times with DEPC water, and stored at -70°C as a template. Use the method of the present invention and the conventional PCR method for sensitivity detection respectively, take 2 μL of DNA dilutions of each concentration after 10-fold dilution as a template, add 23 μL of the detection solution prepared in Example 1 for amplification reaction, and the reaction program is 63 ° C, 40min was taken to load 4 μL of the amplified product, and the results showed that the lower limit of detection of the amplification method for detecting different (RGI and RGII) genotypes of Pseudomonas spp. -1 , ( figure 2 Middle), simultaneously take PCR and 4 μ L of amplification product of the present invention to lo...

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Abstract

The invention provides primers for visually detecting ascochyta citrullina of different genetypes and application thereof. The primers comprise a forward inner primer DB17RG-FIP, a reverse inner primer DB17RG-BIP, a forward outer primer DB17RG-F3, a reverse outer primer DB17RG-B3 and a reverse loop primer DB17RG-LB. The primers supply a novel technical platform to wide and visual detection on the ascochyta citrullina of the different genetypes (RG I and RG II) and can be used for quick and visual detection on the ascochyta citrullina of the different genetypes (RG I and RG II). The method is quick and visual, accurate diagnosis on the ascochyta citrullina of the different genetypes (RG I and RG II) is achieved, and the important practical significance on early disease warning, scientific medicine applying guiding, cost lowering and environmental pollution reducing is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer for visually detecting different genotypes of Pseudomonas spp. and its application. Background technique [0002] Blight is a fungal disease caused by Ascochytacitrullina Smith, whose asexual generation is Didymelabryoniae, which can occur from the seedling stage to the harvest stage of melons. The disease mostly occurs at the base and internodes of the plant stems, causing the entire plant to dehydrate and wither and die after the onset. The host range of vine blight is wide, and it can harm a variety of economic crops such as melon, cucumber, watermelon, and pumpkin in the Cucurbitaceae family. Winter and spring sunlight greenhouses and greenhouse cultivation in early spring and after autumn all occur. In production, the mortality rate of plants can reach 30% to 40%, resulting in serious production reduction. At present, the degree of damage is much higher than that of me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 姚协丰羊杏平李苹芳徐锦华张曼刘广任润生
Owner JIANGSU ACAD OF AGRI SCI