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Detection target of Phytophthora sojae and its pcr primer composition and application

A primer composition and a technology for rot bacteria are applied in the field of genetic engineering to achieve the effects of being easy to popularize and apply on a large scale and having excellent interspecies specificity.

Active Publication Date: 2016-01-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The method of detecting pathogenic bacteria based on PCR has been successfully used in various import and export ports in my country, but the previous target selection was based on the ribosomal gene sequence, but because the ribosomal sequence does not have enough sites to distinguish all pathogenic bacteria, so The development of new detection targets has become a hot spot for detection

Method used

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  • Detection target of Phytophthora sojae and its pcr primer composition and application
  • Detection target of Phytophthora sojae and its pcr primer composition and application
  • Detection target of Phytophthora sojae and its pcr primer composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A common PCR detection kit for detecting Phytophthora rot of soybean, comprising: 20mM upstream primer FP, 20mM downstream primer RP, 10xPCR reaction buffer (TAKARA), 25mMMgCl2 (TAKARA), 2.5mMdNTPMixture (TAKARA), 1.25 units of Taq enzyme (TAKARA), adding ultrapure water to prepare a detection solution.

Embodiment 2

[0029] Example 2 Specificity Test of Phytophthora spp.

[0030] In order to verify the specificity of the common PCR method, the standard strain of Phomopsis sojae (CBS100.87) was selected from different species (Ph. (soybean rust; colloid anthracnose; flathead anthracnose; blast fungus; alternaria; nigrosporium; DNA was used as a template. Take 1 μl of DNA solution, add 23 μl of the detection solution described in Example 1 and 1 μl of sterilized deionized water to carry out ordinary PCR reaction. The reaction program is: 94°C pre-denaturation for 5 minutes; Annealing at °C for 30 sec, extension at 72°C for 45 sec, a total of 35 cycles; finally extension at 72°C for 10 min. The amplified products were subjected to agarose gel electrophoresis, and the results were detected under ultraviolet light. The results show that the specific primers can specifically recognize Phomopsis spp., and samples containing Phytophthora spp. will have a band of about 226bp, while other species ...

Embodiment 3

[0031] Example 3 Intraspecific test of soybean stem rot fungus

[0032] In order to determine whether the method can specifically identify different Phomopsis rot bacteria strains, select the standard bacterial strain DNA of Phomopsis soybean and 14 strains of DNA of Phomopsis soybean strains to be detected as templates, Take 1 μl of DNA solution, add 23 μl of the detection solution described in Example 1 and 1 μl of sterilized deionized water for ordinary PCR reaction, the reaction program is: 94°C pre-denaturation for 5 minutes; then enter the cycle, 94°C denaturation for 30 sec, 60°C annealing for 30 sec, Extend at 72°C for 45 sec, 35 cycles in total; finally extend at 72°C for 10 min. Amplified products were subjected to agarose gel electrophoresis, and the results were detected under ultraviolet light ( figure 2 ).

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Abstract

The invention relates to the field of genetic engineering, and discloses a Phomopsis longicolla Hobbs detection target, a PCR primer composition of the detection target, and applications of the detection target and the PCR primer composition. A nucleotide sequence of the detection target sequence Tef is as shown in SEQ ID NO. 1. An upstream primer and a downstream primer of the common PCR primer composition specific for the detection target are as shown in SEQ ID NO. 2 and SEQ ID NO. 3 respectively. The detection system provided by the invention can detect the Phomopsis longicolla Hobbs quickly with high efficiency, specificity and sensitivity under ordinary PCR amplification conditions, substantially satisfies the urgent needs for detecting the Phomopsis longicolla Hobbs currently, and provides a novel technical platform for Phomopsis longicolla Hobbs detection. The detection system can be used for on-site detection of field quarantine and import and export quarantine, and is easy to be promoted for wide applications.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a detection target of soybean stem rot pathogen and its PCR primer composition and application. Background technique [0002] Phomopsis longicolla Hobbs infects soybeans, causing soybean root rot and seedling disease, and can lead to reduced soybean seed quality [1,2] ,. Soybean stem rot was first discovered in the United States in 1976. Since then, it has been reported in Canada, Argentina, South Korea, Italy, and Yugoslavia. [3,4] . According to statistics, in 1994, the world lost as much as 186,000 tons of soybean production due to this disease [5,6,7] . Because of its widespread occurrence in foreign countries in recent years, large economic impact, high risk of introduction, and no reports of occurrence in my country, it is listed by relevant experts as one of the seven dangerous fungal diseases on soybeans, including Phytophthora soybean [8] . In order to prevent the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/04C12R1/645
Inventor 郑小波沈浩戴婷婷王源超张海峰
Owner NANJING AGRICULTURAL UNIVERSITY