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Detection kit and detection method for flavobacterium columnare

A technology of Flavobacterium columnar and detection kit, which is applied in the direction of microorganism-based methods, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem that there is no quantitative PCR research report for Flavobacterium columnar, and achieve the protection of fish Healthy, easy-to-use procedures, high sensitivity and specificity

Active Publication Date: 2015-11-18
INST OF AQUATIC LIFE ACAD SINICA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant research report on quantitative PCR detection technology for the detection of Flavobacterium columnar in China

Method used

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  • Detection kit and detection method for flavobacterium columnare
  • Detection kit and detection method for flavobacterium columnare
  • Detection kit and detection method for flavobacterium columnare

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0028] Example 1: Primer design and screening for fluorescent quantitative PCR detection of Flavobacterium columnar gyrB gene

[0029] 1. Bioinformatics method to design primers and perform primer screening

[0030] Download the full sequence of the gyrB gene of Flavobacterium columnar in GenBank (GeneBank: CP003222.2), and download the full sequence of the gyrB gene of the negative control bacteria. The negative control bacteria include other species of Flavobacterium, Aeromonas, and Pseudomonas See the table below for genera and related species of Vibrio. After comparison by ClustalX, select the appropriate region to design primers. This region is conserved in Flavobacterium columnar species, but there is at least 10%-30% difference compared with the negative control. After selecting the region, use ABIPrimerExpress3.0 real-time fluorescent quantitative PCR Primer design software to design synthetic primers. The primers allow 2 or less degenerate bases at the same variable...

Embodiment 2

[0068] Embodiment 2: Optimization of the amount of fluorescent quantitative PCR primers

[0069] 1. The first optimization of the amount of fluorescent quantitative PCR primers

[0070] With diluted L1 (10 -4 ) and L2 (10 -5 ) The positive control DNA is used as a template for optimizing the amount of primers, and the upstream and downstream amounts of the primers are optimized by serial dilution within the range of 5.0-12.5 pmol. (5U) 0.6uL, real-time fluorescent PCR buffer (2×) 12.5uL (10×buffer2.5uL of Takara Company of 2.5uL, 2uL of 10mMdNTP and the mixture of 10000×SYBRgreenI0.0025uL and 8uLDEPC water are prepared), Make up to 25uL with sterile water. The PCR reaction conditions were as follows: anti-pollution at 37°C for 5 minutes; then pre-denaturation at 95°C for 3 minutes; final amplification at 95°C for 10 sec and 60°C for 40 sec, a total of 40 cycles, and fluorescent signal detection was performed at the end of each cycle of extension. The results are shown in T...

Embodiment 3

[0081] Example 3: Fluorescent quantitative PCR detection of Flavobacterium columnar gyrB gene

[0082] 1. Establishment of standard curve

[0083] 1.1 Use the positive control plasmid DNA as a template for fluorescent quantitative PCR detection and establish a standard curve. The specific operation is as follows: the plasmid DNA was serially diluted 10 times to I: 1.0×10 10 Copies / uL; II: 1.0×10 9 Copies / uL; III: 1.0×10 8 Copy / uL; Ⅳ: 1.0×10 7 Copy / uL; V: 1.0×10 6 Copy / uL; VI: 1.0x10 5 Copy / uL; VII: 1.0×10 4 Copy / uL; Ⅷ: 1.0×10 3 Copy / uL; IX: 1.0×10 2Copy / uL; X: 1.0×10 1 copy luL;XI: 1.0×10 0 copy / uL. Each dilution was repeated 3 times in parallel. Standard product detection PCR reaction system is: upstream primer 1.5ul (5pmol / ul), downstream primer 2.5ul (5pmol / ul), plasmid DNA 5uL, TaqDNA polymerase (5U) 0.6uL, real-time fluorescent PCR buffer (2×) 12.5 uL (prepared with 10×buffer2.5uL purchased from Takara, 2uL of 10mMdNTP and 10000×SYBRgreenI0.0025uL and 8uLDEPC...

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Abstract

The invention discloses a detection kit and detection method for flavobacterium columnare. The kit contains a pair of primers: an upstream primer GyrB-II-F and a downstream primer GyrB-II-R, designed by taking a GryB gene of the flavobacterium columnare as a target gene. The method can be used for fast and accurately detecting whether an alepidote body or a culturing water body contains pathogenic bacteria or not and achieves targeted fish disease prevention and control.

Description

technical field [0001] The invention relates to the detection field of fish pathogenic bacteria, to a detection kit and detection method of fish pathogenic bacteria, and in particular to a detection kit and detection method of Flavobacterium columnar. Background technique [0002] Flavobacterium columnare, also known as columnar cellophage, is a common fish pathogenic species, mainly harming the gills of fish, and can also cause surface ulcers. At present, there are many detection and diagnostic techniques for this bacterium, including selective medium differential culture method, enzyme-linked immunosorbent assay (ELISA), dot enzyme-linked immunosorbent assay (DOT-ELISA), monoclonal antibody technology (Monoclonal Antibody Technique) , Ordinary PCR (Polymerase Chain Reaction) electrophoresis detection method. These methods have their own characteristics, but they often show shortcomings such as long time-consuming, low precision, poor sensitivity and specificity in product...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/20
CPCC12Q1/6851C12Q1/689C12Q2561/113C12Q2563/107
Inventor 邹红熊凡张金李文祥吴山功王桂堂
Owner INST OF AQUATIC LIFE ACAD SINICA
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