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Aeromonas salmonicida SYBR Green I real-time quantitive PCR (Polymerase Chain Reaction) detection method and application thereof

A technique for auxiliary detection of Aeromonas salmonicida, applied in the biological field, can solve the problems that PCR technology is difficult to achieve high specificity, stable repeatability, and difficult to popularize

Active Publication Date: 2017-03-22
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of Aeromonas salmonicida and its subspecies, ordinary PCR technology is difficult to achieve the high specificity and stable repeatability required for detection, making it difficult to popularize in practical applications

Method used

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  • Aeromonas salmonicida SYBR Green I real-time quantitive PCR (Polymerase Chain Reaction) detection method and application thereof
  • Aeromonas salmonicida SYBR Green I real-time quantitive PCR (Polymerase Chain Reaction) detection method and application thereof
  • Aeromonas salmonicida SYBR Green I real-time quantitive PCR (Polymerase Chain Reaction) detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Design of primers for detection of Aeromonas salmonicida and its detection method

[0040] 1. Design of primers for detection of Aeromonas salmonicida

[0041] According to the sequence of Aeromonas salmonicida fstA gene (accession number: AM712656.1) on GenBank, a pair of specific primers were designed using the primer design software PrimerExplorer and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The primer sequence:

[0042] AsF-F: 5'-TTGTCGGCGAACCTTGTAG-3' (SEQ ID NO: 1);

[0043] AsF-R: 5'-CAAGAGCAAGACGCAGACG-3' (SEQ ID NO: 2).

[0044] 2. Method for detecting Aeromonas salmonicida

[0045] Using the genomic DNA of the fish tissue sample to be tested as a template, the AsF-F / AsF-R primers in step 1 are used to perform fluorescent quantitative PCR to obtain PCR amplification products.

[0046] If the PCR amplification product meets the following conditions: an amplification curve is generated, and the Ct value is 0-38, it means that the samp...

Embodiment 2

[0047] Example 2. Specific detection of primers for detection of Aeromonas salmonicida

[0048] 1. Genomic DNA extraction

[0049] Bacterial Genomic DNA Extraction Kit (purchased from Tiangen Biochemical Technology Co., Ltd.) was used to extract the genomic DNA of the following strains: Aeromonas salmonicida subsp. Aeromonas japonica subspecies, Aeromonas salmonicida subsp. Shirkerii, Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Aeromonas victoria, Ruckeri Yersinia, Edwardsiella tarda, Pseudomonas fluorescens, Staphylococcus aureus, and Micrococcus myelyticus. The number, Latin name and source of each strain are shown in Table 1.

[0050] Table 1 Strains and their sources

[0051]

[0052] 2. Conventional PCR amplification and fluorescent quantitative PCR amplification

[0053] Using the genomic DNA of each strain obtained in step 1 as a template, the primers designed in step 1 were used for conventional PCR amplification and fluorescent quantitative PCR am...

Embodiment 3

[0060] Example 3. Sensitivity detection of primers for detection of Aeromonas salmonicida

[0061] 1. Preparation of plasmid standards

[0062] Measure the concentration and the purity of the solution containing the pMD18T-fstA recombinant plasmid obtained in (1) in 2 of step 2 of Example 2 with a micro-ultraviolet-visible spectrophotometer, and calculate the concentration and purity of the pMD18T-fstA recombinant plasmid per unit volume according to Moore's law. DNA copy number, and the recombinant plasmid as a standard, the calculation formula is as follows:

[0063]

[0064] Among them, the plasmid concentration is 105ng / μL; the plasmid volume is 1μL; the vector length: 2692bp; the fragment length is 170bp. Final calculation result: the DNA copy number contained in each μL recombinant plasmid is 3.3×10 10 .

[0065] Second, the sensitivity of fluorescent quantitative PCR

[0066] The solution containing the pMD18T-fstA recombinant plasmid was subjected to 10-fold ser...

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Abstract

The invention discloses an aeromonas salmonicida SYBR Green I real-time quantitive PCR (Polymerase Chain Reaction) detection method. An aeromonas salmonicida fstA gene is adopted as a target gene, and the aeromonas salmonicida SYBR Green I real-time quantitive PCR detection method is established. An experiment proves that the method provided by the invention not only has the characteristics of high specificity, high sensitivity and good repeatability, but also has the advantages of convenience in operation, quickness in identification, objectivity in results and the like, can be used for quickly diagnosing and quantitively detecting aeromonas salmonicida, and provides a technological means for diagnosis, prevention and control of salmon and bulltrout furunculosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a SYBR Green I real-time quantitative PCR detection method for Aeromonas salmonicida and an application thereof. Background technique [0002] Aeromonas salmonicida was first isolated and described from trout by Emmerich and Weible in 1890. Aeromonas salmonicida have now been identified as non-motile aeromonads of the genus Aeromonas of the family Aeromonadacea. The bacteria can cause furunculosis or canker disease in salmon trout such as Atlantic salmon (Salmo salar), brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), etc. The diseased fish show characteristic abscesses on the side or tail of the body, Ulceration and ulceration may occur in severe abscesses; punctate hemorrhages in the liver and adipose tissue can be observed at autopsy. The bacterium can also be mixed with other bacteria of the genus Aeromonas such as Aeromonas caviae (Aeromonas caviae),...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2561/113C12Q2563/107C12Q2527/101
Inventor 李绍戊刘帅曹永生贾钟贺王荻卢彤岩王渊博杨晨朱国建
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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