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Detection target sequence A3apro of phytophthora sojae, and specific LAMP (loop-mediated isothermal amplification) primer composition and application thereof

A primer composition and the technology of Phytophthora sojae are applied in the field of genetic engineering and achieve the effect of being easy to popularize and apply on a large scale

Active Publication Date: 2012-09-19
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Detection of Phytophthora soybean by PCR technique is based on ribosomal gene sequence [7] , but because the ribosome sequence does not have enough sites to distinguish all pathogenic bacteria, the development of new detection targets has become a hot spot for detection

Method used

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  • Detection target sequence A3apro of phytophthora sojae, and specific LAMP (loop-mediated isothermal amplification) primer composition and application thereof
  • Detection target sequence A3apro of phytophthora sojae, and specific LAMP (loop-mediated isothermal amplification) primer composition and application thereof
  • Detection target sequence A3apro of phytophthora sojae, and specific LAMP (loop-mediated isothermal amplification) primer composition and application thereof

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Experimental program
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Effect test

Embodiment 1

[0037] The results of whole gene sequencing showed that there was a fragment deletion of about 300bp and two small fragment insertions in the Avr3a promoter region of Phytophthora soybean race R7. In the JGI genome database of Phytophthora sojae, there are more than 200 records of the 300bp deletion sequence in the entire gene range of Phytophthora soybean, which are very conserved among each other and distributed in various regions of the genome. The 300bp fragment (SEQ ID NO.1) was determined to be a transposon sequence and named as A3apro. Primers were designed using the online LAMP primer design software primerExplorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html) using A3apro (SEQ ID NO.1) as a template sequence. After uploading the template sequence, the system software calculates and obtains the preliminary LAMP primer combination, and then compares the designed multiple pairs of primers according to the parameters such as the stability of the primers correspondin...

Embodiment 2

[0039] A LAMP detection kit for detecting Phytophthora sojae, comprising: 1.6 μM forward inner primer FIP, 1.6 μM reverse inner primer BIP, 0.2 μM forward outer primer F3, 0.2 μM reverse outer primer B3, 1.4 mM dNTPs, 20mM Tris-HCl pH 8.8, 10mM KCl, 10mM (NH4) 2 SO4, 6mM MgSO 4 , 0.1% Triton X-100, 8U Bst DNApolymerase 320 units, 180mM hydroxynaphthol blue, adding ultrapure water to prepare a detection solution.

Embodiment 3

[0040] The specificity test one of embodiment 3 Phytophthora soybean LAMP reaction

[0041] In order to verify the specificity of the LAMP method, the standard Phytophthora sojae strain 6497 (purchased from ATCC, numbered ATCC 16705, the same below) and different physiological races R3, R6, R8, R12, R14, The DNA of R17, R19, R20, R28, and R31 was used as a template. Take 1 μl of the DNA solution, add 23 μl of the detection solution described in Example 2 and 2 μl of sterilized deionized water to perform the LAMP reaction. The reaction procedure is: 64 ° C for 60 min. The results showed that bands were amplified when LAMP primers were used to amplify the DNA templates of physiological races of Phytophthora sojae; while the negative control could not amplify the target band. The specific LAMP reaction can specifically amplify a trapezoidal band from the tested Phytophthora sojae strain, indicating that the primer composition has species specificity. At the same time, when the D...

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Abstract

The invention belongs to the field of gene engineering, and relates to detection target sequence A3apro of phytophthora sojae, and a specific LAMP (loop-mediated isothermal amplification) primer composition and application thereof. The nucleotide sequence of the detection target sequence A3apro of phytophthora sojae is shown as SEQ ID NO.1. The specific LAMP primer composition designed for the A3apro comprises a forward interprimer FIP: SEQ ID NO..2, reverse interprimer BIP: SEQ ID NO.3, forward outerprimer F3:SEQ ID NO.4, and reverse outerprimer B3:SEQ ID NO.5. The specific LAMP primer composition can be applied to detection or identification of the phytophthora sojae. The LAMP primer composition for detecting the phytophthora sojae has the advantages of excellent specificity sensitivity, quick amplification, high efficiency and convenience in identification.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a detection target sequence A3apro of Phytophthora soybean and its specific LAMP primer composition and application. Background technique [0002] Phytophthora sojae Kaufmann&Gerdemann (Phytophthora sojae Kaufmann&Gerdemann) infecting soybeans causes Phytophthora root rot is one of the devastating diseases in soybean production, and it is also a type of quarantine object announced by my country. Soybean Phytophthora root rot was first discovered in Indiana, USA in 1948 [3] Since then, more than 20 soybean-producing countries such as Canada, Brazil, and Argentina have reported the occurrence of the disease [2，6] . Su Yanchun [10] Soybean Phytophthora root rot was reported for the first time in 1993 in the main soybean producing area of ​​Heilongjiang in my country. Since then, the hazards of Phytophthora soybean have been found in the main soybean producing areas in northern my ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 王源超戴婷婷董莎萌郑小波
Owner NANJING AGRICULTURAL UNIVERSITY
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