Duplex fluorescent quantitation RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for shellfish Bonamia and Perkinsus
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A baglamemia, double fluorescence technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of high requirements and complexity of reagents and primers
Active Publication Date: 2014-03-19
GUANGXI VETERINARY RES INST
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However, it is much more complicated to establish a multiplex fluorescent PCR method than a single-plex one, and it has higher requirements on reagents and primers. At the same time, it is necessary to ensure that there is no mutual interference between the fluorescent groups labeled by different probes. The fluorescent quantitative PCR instrument used has Corresponding Multiple Detection Channels
[0005] At present, there is no report on the detection and diagnosis of shellfish Bolamia and Pichenia using multiplex fluorescent quantitative PCR technology
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Embodiment 1
[0044] Embodiment 1, design and synthesis of primers and Taqman probes
[0045] Two pairs of specific primers and two Taqman probes were designed using the Primer Express 3.0 software according to the conserved sequences of Lammia and Pijenia in GenBank (Table 1).
[0046] Table 1 is the primer and TaqMan probe sequence (5'-3')
[0049] 1. Determination of fluorescent quantitative RT-PCR detection method
[0050] 1. Sample preparation
[0051] According to the instructions of the marine animal tissue genome extraction kit, extract the genomic DNA of Lammia, Monospora, Pichet, Malteia refraction, Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio riverina.
[0052] 2. Preparation of standard products
[0053]The genomic DNA of the above-mentioned obtained Bolamia worm was used as a template for PCR amplification, and the reaction system was 50 μL (containing 25 μL PCR Mix, 1 μmol / L primers (Bonamia (760)-1 and Bonamia (760)-2, see Table 2) ), 19μLddH 2 (0, 4 μL DNA template), the reaction conditions were: 95°C pre-denaturation for 5 min, 94°C for 60 s, 52°C for 60 s, 72°C for 60 s, 35 cycles, and 72°C for 10 min. Obtain 760bp PCR product, this PCR product is cloned into pMD-18T carrier, obtains Bonamia760 plasmid; Sequencing Bo...
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Abstract
The invention discloses a duplex fluorescent quantitation RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for shellfish Bonamia and Perkinsus. The invention provides a primer group for detecting duplex fluorescent PCR of the shellfish Bonamia and the Perkinsus, which consists of a primer 1, a primer 2, a primer 3 and a primer 4. Nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 respectively and sequentially are a sequence 1, a sequence 2, a sequence 4 and a sequence 5 in a sequence table. Experiments prove that the primers and the method which are provided by the invention have the characteristics of short detection time, high sensitivity and strong specificity.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a dual fluorescent quantitative RT-PCR detection kit for shellfish Lammia and Pijenia. Background technique [0002] Bonamia is a major disease that seriously harms the shellfish farming industry, and Bonamiasis is one of the must-report shellfish infectious diseases stipulated by OIE. The parasite parasitizes in the hemolymphocytes of shellfish and can cause considerable prevalence and mortality. Perkinsus (Perkinsus) can cause the shell to fail to close, the mantle to shrink, gonad development to be inhibited, growth to be slow, and abscess to occur occasionally, and the mortality rate can be as high as 95%. These two protozoa are the most susceptible diseases of shellfish and have a very high fatality rate. What's more serious is that people may experience symptoms such as vomiting and diarrhea after eating shellfish infected with Pietia worms. Therefore, it is urgent to establis...
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