LAMP (loop-mediated isothermal amplification) detection primer of fusarium proliferatum and application of LAMP detection primer

A technology for detection of Fusarium exfoliates and primers, which is applied in the fields of identification and control, and detection of crop diseases, can solve the problems of research reports on the rapid detection of Fusarium exfoliates, and achieve accurate and reliable detection results, strong specificity, and detection short cycle effect

Pending Publication Date: 2018-10-19
SHANXI AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] There is no research report on the u...

Method used

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  • LAMP (loop-mediated isothermal amplification) detection primer of fusarium proliferatum and application of LAMP detection primer
  • LAMP (loop-mediated isothermal amplification) detection primer of fusarium proliferatum and application of LAMP detection primer
  • LAMP (loop-mediated isothermal amplification) detection primer of fusarium proliferatum and application of LAMP detection primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The design of the specific primer composition for the LAMP detection of Fusarium exfoliates: using Clustal X software, through the Fusarium exfoliates and other Fusarium ( Fusarium spp.) EF-1alpha Gene sequences were compared and analyzed, and a set of nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.6 was designed using the online LAMP primer design software Primer Explorer V5 software (http: / / primerexplorer.jp / lampv5e) The layered Fusarium-specific LAMP primer composition is specifically composed of a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP and a pair of loop primers Loop F / Loop B.

[0054] The specific primer sequences are shown below.

[0055] F3: 5'-AGTACCAGTGATCATGTTCTTG-3'.

[0056] B3: 5'-TCCTGTCCACAACCTCAA-3'.

[0057] FIP: 5'-AAGTTCGAGACTCCTCGCTACTGAGGAAGTAGGATGAGGTATGA-3'.

[0058] BIP: 5'-CTCGGCCTTGAGCTTGTCAACAATAGGAAGCCGCTGAG-3'.

[0059] Loop F: 5'-TCACCGTCATTGGTATGTTGT-3'.

[0060] Loop B: 5'-AGGCGTACTTGAAGGAACC-3'.

Embodiment 2

[0062] Genomic DNA of the Fusarium pathogenic strains was extracted using the Biospin Fungal Genomic DNA Extraction Kit. The specific extraction process is as follows.

[0063] 1) Select the exfoliated Fusarium pathogenic bacteria cultured on PDA medium for 7-10 days, pick fresh mycelia, grind them into powder, and put them into a 1.5mL centrifuge tube.

[0064] 2) Add 500μL LE Buffer and mix well.

[0065] 3) Incubate at 65°C for 60 minutes (mix by inverting once every 15 minutes), and then remove.

[0066] 4) Add 130 μL DA Buffer, mix well, and bathe in water at 65°C for 5 minutes.

[0067] 5) Centrifuge at 14,000×g for 3 minutes.

[0068] 6) Take the supernatant and transfer it to a new 1.5mL centrifuge tube.

[0069] 7) Add 500μL E Binding Buffer and mix well.

[0070] 8) Transfer the mixture to the Spin Column and centrifuge at 6,000×g for 1 min.

[0071] 9) Add 500μL G Binding Buffer to the Spin Column, centrifuge at 10,000×g for 30s, and discard the liquid in the ...

Embodiment 3

[0076] Based on the LAMP primer composition designed in Example 1, the DNA extracted in Example 2 was used as a template to establish a LAMP detection reaction system for Fusarium sp.

[0077] The LAMP detection reaction system includes 0.2 μM each of the outer primers F3 and B3, 0.8 μM each of the inner primers FIP and BIP, 0.4 μM each of the loop primers Loop F and Loop B, 2×LAMP PCR isothermal amplification reaction mixture (universal type, Sangon Bioengineering (Shanghai) Co., Ltd.) 12.5μL, 8U Bst 0.5 μL of polymerase, 1.0 μL of DNA template, made up to 25 μL with sterilized ultrapure water.

[0078] Preliminarily set the reaction temperature of the LAMP reaction at 62° C. and the time for 30 minutes.

[0079] After the reaction, 2.0 μL of the LAMP amplification product was taken for 2.0% agarose gel electrophoresis detection. The electrophoresis detection conditions were voltage 80V, current 110A, and detection time 30 min.

[0080] If there is a trapezoidal band in the...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) detection primer of fusarium proliferatum and application of the LAMP detection primer. The detection primer is a composition consisting of a pair of outer primers F3/B3, a pair of inner primers FIP/BIP and a pair of loop primers Loop F/Loop B. The LAMP detection primer composition is used for detecting the fusarium proliferatum and detecting whether maize crops are infected with maize kernel rot or not. The designed LAMP detection primer composition of the fusarium proliferatum is high in specificity and good in amplification effect; a detection method has no need of performing separation, purification and culture on pathogenic bacteria, is simple, convenient and fast and is high in practicability, high in sensitivity and accurate and reliable in result; and the LAMP detection primer can be used for early diagnosis of the pathogenic primary stage and early stage of the fusarium proliferatum in maize plant tissues.

Description

technical field [0001] The invention belongs to the technical field of detection, identification and prevention of crop diseases, and relates to a LAMP detection primer for Fusarium laminarum that can cause corn ear rot, a detection kit containing the detection primer, and the use of the detection primer or detection reagent Cartridge LAMP detection method. Background technique [0002] Maize ear rot is one of the most common diseases in the late growth period of maize, which commonly occurs in maize planting areas, with an incidence rate of 5-10%. The incidence rate of susceptible varieties is as high as 50%, even up to 100% in some serious plots, causing serious losses. [0003] Corn ear rot is caused by a variety of pathogens, of which Fusarium is the main pathogen. Fusarium can produce a variety of toxins during the pathogenic process, which can promote cell apoptosis, cause various pathological damages to animal and plant organisms, and bring serious hidden dangers to...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107C12Q2537/1376
Inventor 王燕王春伟王建明张作刚王美琴李新凤郝晓娟
Owner SHANXI AGRI UNIV
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