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A kit and reagent technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems such as the establishment of BonamiaLAMP visual detection kit, etc., to achieve high specificity and improve amplification. High efficiency and high sensitivity
Active Publication Date: 2013-02-27
GUANGXI VETERINARY RES INST
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[0004] At present, there are no related reports on the establishment
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Embodiment 1
[0030] Embodiment 1, the design of primer
[0031] According to the conserved gene sequence of Lammia spp. in GenBank, LAMP primers were designed using the online software Primer Explorer V4 (http: / / primerexplorer.jp / e / v4-manual / index.html). Primers were synthesized by Guangzhou Invitrogen Company. The sequences of the 6 primers are listed in Table 1.
[0032] Table 1 is the LAMP primer sequence
[0033]
[0034] The addition of LF and LB primers can also improve the sensitivity of detection.
Embodiment 2
[0035] Embodiment 2, the application of primer in LAMP detection package Lami worm
[0036] 1. Extraction of nucleic acid
[0037] 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.
[0038] 2. Optimizing the LAMP reaction system, reaction conditions and kit construction
[0039] 25μL LAMP reaction system: 1-4μL dNTPs (10mmol / L, final concentration 0.4mmol / L-1.6mmol / L), 2.5μL 10×Bst buffer, 1μl Bst DNA polymerase 8U (final concentration 320U / L), 4 -7μL Betaine (5mmol / L, the final concentration is 0.8mmol / L-1.4mmol / L), 2-9μL MgSO 4 (25mmol / L, the final concentration is 2mmol / L-9mmol / L), 1μL primers (FIP40μmol / L, BIP40μmol / L, LF20μmol / L, LB20μmol / L, B35μmol / L, F35μmol / L; the final concentration is FIP1.6μmol / L, BIP1.6μmol / L, LF0.8μmol / L, LB0.8μmol / L, B30.2μmol / L, F30.2μmol / L), 1ul Calcein (625μmol / L, the f...
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Abstract
The invention discloses a Bonamia LAMP (loop-mediated isothermal amplification) detection kit. The invention provides a loop-mediated isothermal amplification primer group for detecting Bonamia, and the primer group comprises a primer 1, a primer 2, a primer 3 and a primer 4, wherein the nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are respectively and sequentially sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table. Proven by an experiment, six primers aiming at eight special regions are designed according to the conserved sequences of MDPV (muscovy duck parvovirus), and the six primers are used for carrying out LAMP detection and have the advantages of being more special and more sensitive than a conventional detection method, only one water bath kettle with controllable temperature is used, and the result can be judged by eyes without instruments; and the detection kit is suitable to rapid detection in primary veterinary stations and on livestock farms and has a good application prospect.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for Lammia LAMP. Background technique [0002] Bonamia is the main pathogen affecting the development of shellfish farming in the world. All kinds of shellfish and shellfish of other shellfish families are considered to be susceptible. It mainly parasitizes in blood lymphocytes and can cause severe large death rate. Because of the high pathogenicity and wide geographical distribution of Bonamiasis, Bonamiasis is one of the must-report aquatic animal diseases stipulated by OIE. [0003] Traditional detection methods require the use of expensive instruments and reagents. Loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) is a new nucleic acid amplification technology established by Notomi et al. in 2000. This technology has the advantages of simple operation, fast response, low cost and visualized results. At present, it is widely u...
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