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Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof

A primer composition, Phytophthora soybean technology, applied in the field of genetic engineering, to achieve high accuracy, good practicability, and increase the effect of application value

Active Publication Date: 2012-08-22
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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  • Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof
  • Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof
  • Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A LAMP detection kit for detecting Phytophthora sojae, consisting of 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, 0.2 μM reverse Loop Primer LB, 1.4mM dNTPs, 20mM Tris-HCl pH 8.8, 10mM KCl, 10mM (NH4) 2 SO4, 6mM MgSO 4 , 0.1% Triton X-100, 320 units of 8UBst DNA polymerase, and 180 mM hydroxynaphthol blue were added into ultrapure water to prepare a detection solution.

Embodiment 2

[0037] The specificity test one of embodiment 2 Phytophthora soybean LAMP reaction

[0038] 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 prepared in Example 1 and 2 μl of sterilized deionized water to carry out the LAMP reaction. The reaction procedure is: 64 ° C for 80 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. At the same time, based on the color reaction of the reaction system as the result judgment standard, when amplifying the DNA template of the standard Phytophthora sojae strain 6497 and the physiological ra...

Embodiment 3

[0039] The specificity test two of embodiment 3 Phytophthora soybean LAMP reaction

[0040] In order to verify the specificity of the LAMP method, different species of Phytophthora sojae (Phytophthora ramie; Phytophthora oak; Pythium; Fusarium equisetum; Anthracnose flat head; Magnaporthe grisea; Rhizoctonia solani; Verticillium dahliae) DNA as a template, take 1 μl DNA solution, add 23 μl of the detection solution prepared in Example 1 and 1 μl of incineration Bacterial deionized water was used for LAMP reaction, and the reaction procedure was: 64°C for 80min. The results showed that when the LAMP primers were used to amplify the DNA template of Phytophthora sojae, bands were amplified; however, bacteria of different species and genus from Phytophthora sojae and the negative control could not amplify the target band; At the same time, based on the color reaction of the reaction system as the result judgment standard, when the DNA template of Phytophthora sojae is amplified, ...

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Abstract

The invention belongs to the field of a genetic engineering, and relates to a loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and an application thereof. The primer composition comprises a forward inner primer FIP showed in SEQ ID NO.1, a backward inner primer BIP showed in SEQ ID NO.2, a forward outer primer F3 showed in SEQ ID NO.3, a backward outer primer B3 showed in SEQ ID NO.4, and a reverse annular primer LB showed in SEQ ID NO.5. The LAMP primer composition can be applied to detection or identification of phytophthora rot. The LAMP primer composition has the advantages of good sensibility for specificity, rapid amplification, high efficiency and simple identification in the application of detecting phytophthora rot.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a LAMP primer composition for detecting Phytophthora soybean and its application. Background technique [0002] Phytophthora root rot caused by Phytophthora sojae Kaufmann&Gerdemann infecting soybeans 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 country. Global soybean production currently costs more than $1 billion...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/845
Inventor 王源超戴婷婷董莎萌郑小波
Owner NANJING AGRICULTURAL UNIVERSITY
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