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PCR detection primer and method of Vibro Splendidus and application of PCR detection primer

A technology for detection of Vibrio resplendents and primers, which is applied in the field of detection of Vibrio resplendents, can solve the problems of non-disclosure and achieve fast detection speed, low semi-quantitative detection, and easy observation

Active Publication Date: 2015-11-11
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no publication at home and abroad about the use of Vibrio splendidus fur The DNA sequence of the gene is designed to design a specific nucleic acid identification sequence for ordinary PCR, and relevant research reports on the detection of Vibrio resplendent

Method used

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  • PCR detection primer and method of Vibro Splendidus and application of PCR detection primer
  • PCR detection primer and method of Vibro Splendidus and application of PCR detection primer
  • PCR detection primer and method of Vibro Splendidus and application of PCR detection primer

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specific Embodiment 1

[0023] vibrio brilliant fur The DNA sequence of the gene is similar to that of other Vibrio fur DNA Sequence Alignment of Genes

[0024] Will Vibrio splendidus Vs fur The DNA sequence of the gene ( figure 1 , DNAMAN1) and other Vibrios obtained from NCBI, including Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi fur The DNA sequence of the gene was analyzed for DNA similarity in the "Multiple Sequence Alignment" module in the DNAMAN software, and the results were as follows figure 1 shown. fur Vs The homology of the nucleic acid sequence with Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi is only about 78%, so it can be used as a specific label for the detection of Vibrio splendidus.

specific Embodiment 2

[0025] Design of primers for PCR detection of Vibrio resplendent and establishment of PCR method

[0026] Primers were designed using Primer 5.0 to obtain PCR detection primers VSFurRTF3: 5'-ACAACAACCAGATTGCCAACA-3' and VSFurRTR3: 5'-GATAACTTCACCGCAGTCTAAACAT-3' for detection of Vibrio resplendent. Among them, VSFurRTF3 is located at a position with very low homology, so it can be used to specifically detect Vibrio splendidus. VSFurRTF3 and VSFurRTR3 were used as forward primer and reverse primer respectively, and in 25 μL of PCR reaction system, the following substances were added to make PCR reaction mixture: 2.5 μL of 10×PCR buffer (200 mmolL -1 Tris–HCl (pH8.4), 200mmolL -1 KCl, 15mmolL -1 MgCl 2 ), 0.5 μL of 10 μmolL -1 Forward primer, 0.5 μL of 10 μmolL -1 Reverse primer, 2 μL of dNTP mixture (concentrations of dATP, dGTP, dCTP and dTTP were 2.5 mmolL -1 ), 1 μL of template, 0.25 μL of TaqDNA polymerase, and 18.25 μL of sterile double-distilled water. The reaction...

specific Embodiment 3

[0028] Vibrio brilliant PCR detection primers are used to detect Vibrio brilliant in seawater, the specific steps are as follows:

[0029] (1) After the seawater is filtered and sterilized, the PCR method established in the above specific example 2 is used to detect Vibrio splendidus. The results showed that the detection of Vibrio splendidus in seawater by this method was negative. Therefore, the standard method of Vibrio splendidus was used to detect Vibrio splendidus in seawater;

[0030] (2) Will grow to OD 600 About 0.5 Vibrio splendidus, diluted by 10-fold concentration gradient to the final concentration of 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 and 1.0×10 0 CFUmL -1 Take 1 μL of seawater as the template for the PCR reaction;

[0031] (3) To 25 μL of PCR reaction system, add the following volumes of substances: 2.5 μL of 10×PCR buffer (200mmolL -1 Tris–HCl (pH8.4), 200mmolL -1 KCl, 15mmolL -1 MgCl 2 ), 0.5 μ...

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Abstract

The invention discloses a PCR detection primer and method of Vibro Splendidus and application of the PCR detection primer. The PCR detection primer is characterized by being designed according to the DNA sequences of a fur gene of Vibro Splendidus, the sequences of the designed PCR detection primer are VSFur RTF3:5'-ACAACAACCAGATTGCCAACA-3' and VSFurRTR3:5'-GATAACTTCACCGCAGTCTAAACAT-3'. The PCR detection method includes the following steps of firstly, preparing a matrix of seawater, apostichopus japonicus epidermis, apostichopus japonicus muscle or apostichopus japonicus coelomic fluid; secondly, establishing a 25-microlitre PCR reaction system; thirdly, conducting a PCR reaction, and finally conducting agarose gel electrophoresis to conduct result analysis. The PCR detection primer can be used for detecting Vibro Splendidus in seawater, apostichopus japonicus epidermis, apostichopus japonicus muscle or apostichopus japonicus coelomic fluid and has the advantages of being high in detection speed and sensitivity.

Description

technical field [0001] The invention relates to the detection of Vibrio brilliant, in particular to a primer for PCR detection of Vibrio brilliant and a detection method and application thereof. Background technique [0002] Vibrio splendidus ( Vibriosplendidus ) widely exists in the marine environment. It is a gram-negative bacterium, rod-shaped, movable, and has a polar single flagella. It is divided into biological type I and biological type II. Both are pathogenic and are important pathogenic bacteria of marine animals. One of them can infect hosts such as echinoderms, bivalve molluscs and crustaceans, leading to the occurrence of aquaculture diseases. Therefore, in order to meet the requirements of rapid diagnosis and early warning control of this bacterium in the process of aquaculture, the sensitive and rapid detection of this bacterium is of great significance. [0003] The detection methods of Vibrio resplendent mainly include immunological detection and molecular...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/63
CPCC12Q1/686C12Q1/689C12Q2600/158C12Q2531/113
Inventor 张卫卫梁伟康李成华韩庆喜
Owner NINGBO UNIV
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