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

Pending Publication Date: 2018-02-23
INST OF OCEANOLOGY & MARINE FISHERIES JIANGSU
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional chromogenic medium technology cannot distinguish between normal Vibrio and variant Vibrio, and cannot provide support for the detection of AHPNS pathogens

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to an LAMP (loop-mediated isothermal amplification) test kit for hepatopancreas necrosis disease in Litopenaeus vannamei, comprising the reagents of DNA extracting reagent, a primer, an amplification system reagent and a DNA standard. The LAMP test kit of the invention has the advantages that test on AHPND (acute hepatopancreas necrosis disease) in Litopenaeus vannamei is more efficient and sensitive, effective surveillance is ensured for AHPND pathogens in fry or adults during farming of Litopenaeus vannamei, farming risk is lessened, and the LAMP test kit has a promising application prospect.

Description

technical field [0001] The invention belongs to the field of detection of prawn diseases, in particular to a LAMP detection kit for hepatopancreatic necrosis of Penaeus vannamei. Background technique [0002] Since 2009, Acute hepatopancreas necrosis syndrome (AHPNS) in prawns was first discovered in Hainan, my country. Subsequently, AHPNS broke out in Vietnam, Thailand, Malaysia and the Philippines, and Thailand and other places suffered severe production cuts. Since 2011, the main production areas in southern my country, such as Guangdong and Guangxi, have suffered serious production reductions due to the impact of AHPNS. [0003] After the occurrence of AHPNS in Litopenaeus vannamei, the body color turns white, the shell becomes soft, the food intake decreases significantly, the vitality weakens, and the action is slow. A few prawns have black spots on the body surface; Obvious atrophy or abnormal hypertrophy, jejunum empty stomach. The seedlings can die after 10 days o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844
CPCC12Q1/6888C12Q1/6844C12Q2531/119
Inventor 沈辉乔毅万夕和蒋葛范贤平王李宝史文军黎慧
Owner INST OF OCEANOLOGY & MARINE FISHERIES JIANGSU
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