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Loop-mediated isothermal amplification primer composition for detecting phytophthora infestans and application thereof

A primer composition, Phytophthora infestans technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial determination/inspection, etc.

Active Publication Date: 2018-10-19
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used target genes in common PCR include the ribosomal gene transcription spacer (Internal transcribed space, ITS), but because the ribosomal 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 primer composition for detecting phytophthora infestans and application thereof
  • Loop-mediated isothermal amplification primer composition for detecting phytophthora infestans and application thereof
  • Loop-mediated isothermal amplification primer composition for detecting phytophthora infestans and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Design of LAMP primers

[0034] The selection of new target genes and the screening of primers are the key factors for LAMP detection. According to reports, A3aPro is a highly conserved and high-copy transposon in Phytophthora soybean, which can be used for LAMP detection of Phytophthora soybean. Using the sequence of A3aPro to perform Blast in the Phytophthora infestans genome Broad Institute (http: / / www.broad.mit.edu / ), a 178bp DNA homologous sequence was identified, and then further Blasted with this 178bp sequence, identified A specific high-copy sequence, and named it PiSMC ( P . i nfestans s specific m multiple c opy DNA sequence) (SEQ ID NO.1). Primers were designed using the online LAMP primer design software primerExplorer V5 (http: / / primerexplorer.jp / lampv5e / index.html) using PiSMC (SEQ ID NO.1) as a template sequence. After uploading the template sequence, the system software calculates and obtains the preliminary LAMP primer combination, an...

Embodiment 2

[0036] A LAMP detection kit for detecting Phytophthora infestans, 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, 1.4mM dNTPs, 20mM Tris-HCl pH 8.8, 10mM KCl, 10mM(NH4) 2 SO 4 , 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

[0037] Embodiment 3: the specificity test of Phytophthora infestans LAMP reaction

[0038] To verify the specificity of the LAMP method, the DNA of the standard Phytophthora infestans strain T30-4 and 14 other strains from different geographical regions of Europe, South America and Asia were selected as templates (Table 2). Take 1 μl of DNA solution, add 23 μl of LAMP detection solution and 1 μl of sterilized deionized water for LAMP reaction, the reaction program is: 64°C for 70 minutes. Results According to the color change in the reaction system, it was judged whether Phytophthora infestans could be detected. The results of reactions containing P. infestans DNA (HNB showing the reaction) were all blue, while the negative control was purple. This shows that the LAMP detection method established by the present invention has good versatility ( figure 2 ). In addition, 31 plant pathogens were selected to further test their specificity. These 31 pathogens came from 18 genera...

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Abstract

The invention belongs to the field of genetic engineering and discloses a loop-mediated isothermal amplification primer composition for detecting phytophthora infestans and application thereof. A detection target of the phytophthora infestans is PiSMC; the nucleotide sequence of the phytophthora infestans is shown as SEQ ID NO. 1; a forward internal primer FIP of a specific LAMP primer compositionof the target sequence is shown as SEQ ID NO. 2; a reverse internal primer BIP is shown as SEQ ID NO. 3; a forward external primer F3 is shown as SEQ ID NO. 4; a reverse external primer B3 is shown as SEQ ID NO. 5; a reverse loop primer LB is shown as SEQ ID NO. 6. The detection system is capable of rapidly and efficiently detecting the phytophthora infestans with high specificity and high sensitivity under the LAMP amplification condition; meanwhile, the detection results can be observed by naked eyes; therefore, the detection system has a great value of application in detection of late blight of potatoes in the field.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a loop-mediated isothermal amplification primer composition for detecting potato late blight and its application. Background technique [0002] Potato late blight caused by Phytophthora infestans is a devastating plant disease, causing losses of more than $6 billion worldwide annually. In the middle of the 19th century, late blight broke out in European planting areas, and Ireland suffered from two consecutive years of disasters, resulting in the failure of potato production, causing hundreds of thousands of people to starve to death, and 1.5 million people to emigrate. As early as 1992, the Center International Potato (CIP) had listed late blight as an important research target. In order to strengthen the research on potato late blight in various countries in the world, in 1996, the CIP headquarters established the International Potato Late Blight Research Collaborative Networ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 孔亮汪慧斌王帅帅徐萍萍张若芳董莎萌郑小波
Owner NANJING AGRICULTURAL UNIVERSITY
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