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A kind of lamp primer for detecting watermelon bacterial fruit spot and its detection method

A fruit spot fungus and bacterial technology, applied in the field of detection of LAMP primers and detection of watermelon bacterial fruit spot fungus, to achieve simple and rapid detection and reduce the possibility of contamination

Active Publication Date: 2021-03-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For watermelon fruit blotch, domestic and foreign researchers have initially established a LAMP detection system, but the specificity and sensitivity of the designed primers still need to be improved, and the LAMP reaction system needs to be further improved

Method used

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  • A kind of lamp primer for detecting watermelon bacterial fruit spot and its detection method
  • A kind of lamp primer for detecting watermelon bacterial fruit spot and its detection method

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Experimental program
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Effect test

Embodiment 1

[0033] Design of specific primers for loop-mediated isothermal amplification (LAMP) detection of watermelon fruit spot disease and validation of primer specificity

[0034] (1) Design of specific primers for LAMP detection of watermelon fruit blotch

[0035] Download the genome sequence of Acidovorax avenae subsp.citrulli and its related species from the NCBI database, conduct homology analysis to find highly conserved and specific genes suitable for designing LAMP primers, and determine the bacterial fruit spot of melons The housekeeping gene ugpB (Accession No. Aave_0609) encoding extracellular lysis binding protein is the target gene of this quarantine pathogen.

[0036] Using the online design software Primer Explorer V4 to design LAMP primers with high specificity, as shown in SEQ ID NO.1-5, the sequence was synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0037] (2) Extraction of genomic DNA of test strains

[0038] Extraction was performed according to the ins...

Embodiment 2

[0046] Sensitivity determination of loop-mediated isothermal amplification (LAMP) detection for watermelon fruit spot disease

[0047] Use the Nanodrop ND-2000 ultra-trace nucleic acid protein analyzer to measure the concentration of genomic DNA of watermelon fruit spot bacteria, and then use double distilled water to carry out 10-fold gradient dilution of genomic DNA (10 -1 ~10 -7 ), and 2 μL of different dilutions of DNA were used as templates for LAMP amplification.

[0048] The result is as figure 2 As shown, the detection sensitivity of the LAMP system for detecting bacterial fruit spot of melons can reach 1.72×10 2 fg / μL.

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Abstract

The invention discloses loop-mediated isothermal amplification (LAMP) primers for detecting watermelon acidovorax citrulli as well as a detection method thereof, and belongs to the field of a biological technology. The LAMP primers comprise two outer primers, two inner primers and one cyclizing primer, wherein the nucleotide sequences are as shown as SEQ ID NO.1 to 5 correspondingly. The LAMP primer provided by the invention has specificity on detection of the watermelon acidovorax citrulli and has detection sensitivity which reaches to 1.72*102 fg / uL; by the detection method provided by the invention, the watermelon acidovorax citrulli can be detected simply and rapidly and the result can be judged by naked eyes; and a color developing agent is added before LAMP reaction, so that the possibility of pollution is greatly reduced, and the LAMP primers are suitable for field on-site detection of grass-roots agricultural departments and related detection departments.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a LAMP primer for detecting bacterial fruit spot of watermelon and a detection method thereof. Background technique [0002] Bacterial fruit spot of melon is the most serious and destructive seed-borne disease in watermelon and melon cultivation. The pathogen is Acidovorax avenae subsp.citrulli (Aac), which is a worldwide quarantine disease. Since the disease was reported in my country in the mid-1980s, it has occurred in most parts of the country one after another, causing great harm, heavy losses, reduced or even no harvest of fruits and vegetables, and a huge threat to production. In recent years, it has become an urgent need for melon production at home and abroad. The outstanding problem solved, the disease has been listed as my country's entry quarantine pests and national agricultural plant quarantine pests. [0003] Existing detection technologies mainly include traditional p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 吴秀芹洪纤纤杨敏姚榕罗金燕陈磊安千里李斌
Owner ZHEJIANG UNIV