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A primer pair for detecting watermelon bacterial fruit spot and its application

A kind of fruit spot fungus and bacterial technology, which is applied to the detection and identification of primer pairs and the detection field of watermelon bacterial fruit spot fungus, can solve the problems of low primer specificity, low versatility, false negatives, etc., and achieve high sensitivity and specificity good sex effect

Active Publication Date: 2022-04-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a kind of method that can accurately identify and quarantine watermelon bacterial fruit spot bacterium, to overcome existing PCR method detection watermelon bacterial fruit spot bacterium existence low specificity or versatility, detection is easy to appear false. Disadvantages of Positives or False Negatives

Method used

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  • A primer pair for detecting watermelon bacterial fruit spot and its application
  • A primer pair for detecting watermelon bacterial fruit spot and its application
  • A primer pair for detecting watermelon bacterial fruit spot and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Design of specific primers for the detection of watermelon bacterial fruit spot

[0036] 1. According to the genome sequence of watermelon bacterial fruit blotch in the NCBI genome database, PGAP was used to conduct pan-genomic analysis on watermelon fruit blotch and its related species, and it was found that all strains of Acidovorax citrulli were present, but other related species were not. The specific marker protein, and then the nucleotide sequence of the coding gene of the specific protein was performed local BLASTN in the database composed of the whole genome sequence of all strains of Acidovorax citrulli, to reconfirm that the sequence only has a high degree of homology within the species, and confirm the registration The protein with the number WP_017438750.1 is a unique protein of Acidovorax citrulli.

[0037] 2. Design primers for the specific protein of Acidovorax citrulli, and evaluate the generality and specificity of the primers to each strain of Acidovor...

Embodiment 2

[0081] Establishment of a molecular detection method for bacterial fruit spot of watermelon

[0082] 1) DNA extraction of the sample to be tested

[0083] Pick a single colony of the strain to be tested in 5mL NA liquid medium, 220r min -1 , shake culture at 30℃ for 12h, until the concentration is 10 8 CFU mL -1 , collected the bacteria by centrifugation, and extracted the bacterial genomic DNA using the TIANGEN Bacterial Genomic DNA Extraction Kit. The UV spectrophotometer Nanodrop 2000 was used to detect the quality and concentration of the extracted DNA, and it was stored at -20°C for later use.

[0084] 2) Establishment of PCR amplification system and method

[0085] The PCR amplification reaction system is a 25 μL system, specifically: 1 μL DNA template, 1 μL upstream primer Aac1F, 1 μL downstream primer Aac1R, 12.5 μL 2×Hieff PCP Master Mix, 9.5 μL ddH 2 O.

[0086] The PCR amplification program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s,...

Embodiment 3

[0089] Specific Detection of Primer Pairs for Bacterial Fruit Spot of Watermelon

[0090] Tested strains: 6 strains of watermelon bacterial fruit spot pathogens, namely: Acidovorax citrulli Aac1, Acidovorax citrulli Aac2, Acidovorax citrulli AH01, Acidovorax citrulli HB02, Acidovorax citrulli SD01, Acidovorax citrulli XJL12, all preserved by our laboratory;

[0091]6 control strains, namely: rice acidophilus Acidovorax oryzae RS1 and Acidovorax oryzae RS2 were preserved by our laboratory, rice acidophilus Acidovorax oryzae CGMCC 1.1728 T , Acidovorax avenae CGMCC 1.1726 T , Acidovorax delafieldiiCGMCC 1.2784 T Purchased from China Common Microorganism Culture Collection and Management Center, soil acidophilus Acidovorax soli DSM25157 T purchased from China Marine Microorganism Culture Collection Management Center.

[0092] Extract the DNA of 6 bacterial fruit spot pathogens of watermelon and 6 reference strains, take 1 μL DNA solution as a template, and use the primer pair ...

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Abstract

The invention discloses a pair of primers for detecting watermelon bacterial fruit spot fungus (Acidovorax citrulli) and an application thereof, belonging to the field of biotechnology. The primer pair includes an upstream primer and a downstream primer, the nucleotide sequences of which are respectively: 5'-TTCACCGACTACACCAGTGC-3'; 5'-CATCAGTTCGTAGTGGCGGT-3'. The primer pair provided by the invention has good specificity and high sensitivity, and can specifically amplify the unique gene fragment of watermelon bacterial fruit spot pathogen. The minimum detectable concentration of DNA of P. fruit spot reached 40pg / μL. The detection method provided by the invention can quickly and accurately identify the watermelon bacterial fruit spot pathogen in the watermelon sample, and has important application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pair of primers and a detection method for detecting and distinguishing Acidovorax citrulli. Background technique [0002] Watermelon is a traditional summer fruit. As the country with the largest planting area of ​​watermelon in the world, my country has always regarded it as the leading product with priority in the adjustment of the planting industry structure. The healthy development of the watermelon industry is promoting farmers’ income, adjusting the stubble layout, and enriching the fruit market. and other aspects play an important role. In recent years, with the improvement of living standards, growers and consumers have paid more and more attention to issues such as the quality of melons and fruits. [0003] Bacterial fruit spot of watermelon was first discovered in the United States in 1987 and has since occurred worldwide. In recent years, more than 20 provinces and auto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/686C12Q2565/125
Inventor 张健男罗金燕张洁净谢婷安千里李斌
Owner ZHEJIANG UNIV
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