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PCR (Polymerase Chain Reaction) detection primer, fluorescent quantitative PCR detection kit and detection method for bychowskyella pseudobagri achmerov

A technology of four anchor worms and detection kits, which are applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as the absence of fluorescent quantitative PCR research reports for the four anchor worms , to achieve the effect of being suitable for large-scale promotion and application, high sensitivity and specificity, and disease prevention

Active Publication Date: 2017-07-25
扬州宏盛水产科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no domestic research report on the detection technology of Tetraanchor chrysalis in China.

Method used

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  • PCR (Polymerase Chain Reaction) detection primer, fluorescent quantitative PCR detection kit and detection method for bychowskyella pseudobagri achmerov
  • PCR (Polymerase Chain Reaction) detection primer, fluorescent quantitative PCR detection kit and detection method for bychowskyella pseudobagri achmerov
  • PCR (Polymerase Chain Reaction) detection primer, fluorescent quantitative PCR detection kit and detection method for bychowskyella pseudobagri achmerov

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1 Primer Design and Screening for Fluorescent Quantitative PCR Detection of the 18s Variable Region Gene of Tetraanchoris chinensis

[0098] 1. Bioinformatics method to design primers and conduct primer screening

[0099] According to the literature information, the variable region of Tetraanchoris chinensis 18s (GenBank accession number: KY680236) screened and obtained by the Institute of Hydrobiology, Chinese Academy of Sciences was analyzed, and the 18s gene sequences of 12 related parasites were downloaded as reference sequences. The following table.

[0100] Reference sequence species name (Latin name) GenBank Mizelleus indicus KR296800 Bychowskyella tchangi KT852455 Bychowskyella fossilisi KT852454 Mizelleus longicirrus KR296801 Hamatopeduncularia elongata KT252896 Thysanotohaptor rex KT252903 Neocalceostomoides spinivaginalis KT252904 Hamatopeduncularia thalassini KT252900 Chauhanell...

Embodiment 2

[0163] Embodiment 2: Optimization of the amount of fluorescent quantitative PCR primers

[0164] 1. The first optimization of the amount of fluorescent quantitative PCR primers

[0165]Use the diluted L1 (20×) and L2 (400×) positive control plasmids as templates for optimizing the amount of primers, and perform gradient optimization on the upstream and downstream amounts of the primers respectively. The concentration of the primer working solution is 10 pmol / μL, and the PCR reaction system is: The amount of upstream and downstream primers is shown in Table 2, sample (template) DNA 5 μL, Taq DNA polymerase (5U) 0.6 μL, real-time fluorescent PCR buffer (2×) 12.5 μL (2.5 μL 10× buffer from ThermoFisher Company 2.5 μL, 2 μL 10mM dNTP and a mixture of 10000×SYBRgreen I 0.0025μL and 8μL DEPC water), supplemented with sterile water to 25μL. The PCR reaction conditions were: 95°C for 3min; 95°C for 10s, 55°C for 20s, 72°C for 20s, 75°C for 5s, and a total of 40 cycles for plate readi...

Embodiment 3

[0177] The establishment of embodiment 3 standard curve

[0178] 1. Fluorescence quantitative PCR detection of positive control plasmid

[0179] The positive control plasmid DNA was used as a template for fluorescent quantitative PCR detection to establish a standard curve. The specific operation is as follows: the plasmid DNA was serially diluted 20 times to 1:3.0×10 10 copies / μL; 2: 1.5×10 9 copies / μL; 3: 7.5×10 7 copies / μL; 4: 3.75×10 6 Copy / μL; 5: 1.88×10 5 copies / μL; 6: 9.38x10 3 copies / μL. Each dilution was repeated 3 times in parallel. Standard detection PCR reaction system is: 2.0 μL (10 pmol / μL) of upstream primer, 2.0 μL (10 pmol / μL) of downstream primer, 5 μL of plasmid DNA, 0.6 μL of Taq DNA polymerase (5U), real-time fluorescent PCR buffer (2× ) 12.5 μL (prepared with 10×buffer 2.5 μL purchased from ThermoFisher, 2 μL of 10 mM dNTP and 10000×SYBRgreen I 0.0025 μL and 8 μL DEPC water mixture), supplemented with sterile water to 25 μL. The PCR reaction cond...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) detection primer, a fluorescent quantitative PCR detection kit and a detection method for bychowskyella pseudobagri achmerov. A special primer for the fluorescent quantitative PCR detection on a 18s variable-region gene of the bychowskyella pseudobagri achmerov, a kit containing the primer and a detection method thereof are provided. The primer comprises an upstream primer 18s-II-F and a downstream primer 18s-II-R; the sequence of the 18s-II-F is 5'-CTGGTCATGGCTCTGCTGAC-3'; the sequence of the 18s-II-R is 5'-CGTTAAAGGAGCATCAGCTG-3'. According to the PCR detection primer, the special primer in the fluorescent quantitative PCR detection of the 18s gene of the bychowskyella pseudobagri achmerov is provided for the first time; the purpose of detecting the 18s variable-region gene in a to-be-detected sample by utilizing fluorescent quantitative PCR is enabled to become possible.

Description

technical field [0001] The invention relates to the field of fish parasite detection, in particular to a PCR detection primer, a fluorescent quantitative PCR detection kit and a detection method for Tetraanchor chrysanthemum. Background technique [0002] Bychowskyella pseudobagri Achmerov, which parasitizes on the gills of yellow catfish, has a wide distribution and has been recorded in Northeast China, Shandong, Zhejiang, Hubei, Guangdong, Jiangsu, Fujian, Yunnan, Sichuan, etc., and has a wide range of hosts. It can parasitize on the gills of a variety of fishes of the genus Pelada and Hemibagrus. Small insect body, body length 0.210-0.588mm, body width 0.060-0.168mm. It belongs to a kind of monogenetic trematode. When it parasitizes the gills of fish, it shows obvious "group living" phenomenon. When it parasitizes a small amount of Tetraanchor chinensis, the cultured fish have no obvious symptoms, and most of them show a decrease in food intake or a dark floating head ph...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2531/113C12Q2563/107C12Q2561/113
Inventor 张秧兆张东王桂堂张露邹红
Owner 扬州宏盛水产科技有限公司
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