LAMP (loop-mediated isothermal amplification) Test kit for hepatopancreas necrosis disease in Litopenaeus vannamei
A detection kit and hepatopancreas technology, applied in the field of LAMP detection kits for hepatopancreatic necrosis of Penaeus vannamei, can solve the problems of being unable to distinguish between normal Vibrio and variant Vibrio, and cannot provide support for AHPNS pathogen detection, and achieve good application Foreground, avoidance of aerosol pollution, effects of increased efficiency and sensitivity
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Embodiment 1
[0025] This embodiment provides a LAMP detection kit for Penaeus vannamei hepatopancreatic necrosis, including the following reagents: DNA extraction reagents, primers, amplification system reagents, DNA standard items (amount of more than 1 time, preferably 10-15 times) consumption); wherein, the specific sequence of the primer is as follows:
[0026] F3: 5'-GGGATCAAGGACGAAGGCT-3';
[0027] B3: 5'-CAGCACAATCCACTCCTGG-3';
[0028] FIP: 5'-AAGCATCGCTCTCGCAACACC-ACACCGCTGTAGTTCTAGCA-3';
[0029] BIP: 5'-TTCGGGCTCTGGGGATAGTACG-GTCCTTCCGTCAATTTCGCT-3'.
[0030] DNA extraction reagents can be conventional DNA extraction reagents, amplification system reagents mainly include dNTPs mixture, betaine, Mg 2+ , BstDNA polymerase, hydroxynaphthol blue dye, etc., each reagent is stored separately. The DNA standard was Hepatopancreatic Necrosis (AHPND) DNA.
[0031] Further, the amplification system reagents include 2mM dNTPs mixture, 0.8M betaine, 6mM Mg 2+ , 0.16 U / μL of BstDNA poly...
Embodiment 2
[0033] This embodiment provides the detection steps of a LAMP detection kit for Penaeus vannamei hepatopancreatic necrosis, specifically as follows:
[0034] 1) Collection of samples of Penaeus vannamei
[0035] Use sterilized equipment to take Penaeus vannamei (200 seedlings are mixed into a sample, and a single adult shrimp is a sample) into sterile sealed bags, refrigerated or frozen and brought back.
[0036] 2) Sample DNA extraction
[0037] Seed samples were directly ground, and hepatopancreas tissues were taken as samples from adult shrimps for grinding, and 30 mg of the ground samples were transferred to a sterilized 1.5 mL centrifuge tube. An extraction kit (Mag-MK Animal Genomic DNA extraction kit, Shanghai Sangong)) was used to extract sample DNA as a template for LAMP amplification.
[0038] 3) Design of primers
[0039] Using PrimerExplorer 4 software, primers were designed using the published hepatopancreatic necrosis (AHPND) ssurRNA gene sequence (GenBank acc...
Embodiment 3
[0045] Sensitivity determination
[0046] 1) Hepatopancreatic necrosis (AHPND) DNA extraction: pick positive samples of hepatopancreatic necrosis (AHPND), and use a commercial DNA extraction kit (Mag-MK Animal Genomic DNA extraction kit (Mag-MK Animal Genomic DNA extraction kit) , Shanghai Sangong)) to extract sample DNA.
[0047]2) Conventional PCR amplification: Amplify the DNA of the positive sample of Hepatosinocida enterica with F3 and B3 primers, the amplification program: 94°C for 5m; 94°C for 30s, 58°C for 30s, 72°C for 45s, cycle 35 times; 72°C for 7m. The amplified product was subjected to 1.5% agarose electrophoresis, and bands with the expected size (about 200 bp) were detected.
[0048] 3) Gel recovery: recover the amplified product obtained in 2) by referring to the AxyPrep DNA Gel Recovery Kit method.
[0049] 4) Carrier connection: refer to the operating instructions of pMD19-T Vector, prepare the following DNA solution in a microcentrifuge tube, the total vo...
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