Degenerate primer group, kit and method for LAMP detection of prawn white spot syndrome virus

A detection kit and technology for white spot syndrome, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as the inability to complete the detection of various white spot syndrome virus strains in shrimp, and achieve fast detection speed, The effect of high sensitivity and easy operation

Pending Publication Date: 2021-05-28
珠海市迪奇孚瑞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The existing LAMP detection primers, kits and detection methods for shrimp white spot syndr

Method used

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  • Degenerate primer group, kit and method for LAMP detection of prawn white spot syndrome virus
  • Degenerate primer group, kit and method for LAMP detection of prawn white spot syndrome virus
  • Degenerate primer group, kit and method for LAMP detection of prawn white spot syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the design of the degenerate primer group that prawn white spot syndrome virus LAMP detects

[0040] According to the Chinese Taiwan strain (TW, AF440570), Thailand strain (TH, AF369029), Chinese strain (CN, AF332093), Korean strain (KR, JX515788) and the largest genome WSSV-TH retrieved from GenBank -96-II (AY753327) The conserved sequence of the five genomes, designed a degenerate primer set for the detection of shrimp white spot syndrome virus LAMP, the degenerate primer set includes 5 specific primers OF, OR, IF, IR and LB, specifically The sequence is as follows:

[0041]OF: 5'-CTTCAGACCATATKTAAGAGTG-3';

[0042] OR: 5'-GCAGATTCCAAACCAGGAA-3';

[0043] IF: 5'-GAGCGGGCATAGAAGTGTAAAAMAT-TCTCTTCTTGGAATGATGAACG-3';

[0044] IR: 5'-TCCAATATGAAGCAATGGGAGCTA-GGTTTAAGCCATGGTCTCT-3';

[0045] LB: 5'-GTGCTAATGGCAGCTGAA-3'.

[0046] A degenerate primer refers to a mixture of primers representing all the different base sequence possibilities encoding a singl...

Embodiment 2

[0049] Embodiment 2, establishment of prawn white spot syndrome virus LAMP detection kit

[0050] The shrimp white spot syndrome virus LAMP detection kit includes the shrimp white spot syndrome virus LAMP detection degenerate primer set in Example 1, LAMP reaction solution, color reagent, positive control group and negative control group.

[0051] The LAMP reaction solution includes 5× constant temperature amplification buffer, dNTP and DNA polymerase; the DNA polymerase is BstDNA polymerase; the chromogenic reagent includes SYBR™ Green I fluorescent dye.

[0052] The positive control group is the T vector containing the ATPase gene fragment of prawn white spot syndrome virus.

[0053] The negative control group was TE buffer.

Embodiment 3

[0054] Embodiment 3, the establishment of the detection method of prawn white spot syndrome virus LAMP

[0055] 1. Extraction of DNA from samples to be tested

[0056] The sample to be inspected in the embodiment of the present invention is shrimp seedling or adult shrimp tissue, and the DNA of the sample to be inspected is extracted by grinding, and the specific operation is as follows:

[0057] 1.1 Take about 100mg of the sample to be tested, put it in a 1.5ml sterilized centrifuge tube, and use a grinding rod to grind the sample to be tested in the centrifuge tube until it is homogenized;

[0058] 1.2 Add 500ul lysate, mix thoroughly; lyse at room temperature for 10 minutes;

[0059] 1.3 Use a centrifuge to centrifuge at 12000g for 1 minute, take the supernatant, and add it to the DNA adsorption column;

[0060] 1.4 Use a centrifuge with a centrifugal force of 8000g for centrifugation, so that the supernatant passes through the adsorption column, so that the viral DNA is ...

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Abstract

The invention discloses a degenerate primer group, a kit and a method for LAMP detection of a prawn white spot syndrome virus. The degenerate primer group comprises a primer OF, a primer OR, a primer IF, a primer IR and a primer LB, the primer OF: 5'-CTTAGACCATATKTAAGAGTG-3'; the primer OR:5'-GCAGATTCCAAACCAGGAA-3'; the primer IF: 5'-GAGCGGGCATAGAAGTGTAAAAMAT-TCTCTTCTTGGAATGATGAACG-3'; the primer IR: 5'-TCCAATATGAAGCAATGGGAGCTA-GGTTTAAGCCATGGTCTCT-3'; and the primer LB: 5'-GTGCTAATGGCAGCTGAA-3'. Specific amplification of the prawn white spot syndrome virus is completed by designing the degenerate primer group for LAMP detection of the prawn white spot syndrome virus, the specificity is good, the sensitivity is high, and an LAMP detection kit for the prawn white spot syndrome virus is designed on the basis. The LAMP detection kit can complete detection of various prawn white spot syndrome virus strains within 40 minutes, is high in detection speed and simple and convenient to operate, and can be widely applied to rapid differential diagnosis of various prawn white spot syndrome virus strains.

Description

technical field [0001] The invention relates to the technical field of biological virus detection, in particular to a degenerate primer set, a kit and a method for detecting prawn white spot syndrome virus LAMP. Background technique [0002] Shrimp white spot syndrome virus (WSSV) is a large baculovirus that can widely infect seedlings, adults, and broodstock of decapod crustaceans such as shrimps and crabs. Cause a large number of deaths of cultured prawns, and cause huge economic losses to the aquaculture industry. Therefore, it is of great significance to detect WSSV quickly, accurately and sensitively, strengthen the quarantine of seedlings and adult shrimps, and monitor the environment of aquaculture water bodies to prevent virus infection, cut off virus transmission, and ensure the healthy and sustainable development of my country's shrimp aquaculture industry. [0003] At present, there are six main detection methods for prawn white spot syndrome virus (WSSV): (1) El...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2521/101C12Q2563/107
Inventor 董铖梁美贤陈天蓝罗达圣唐翔
Owner 珠海市迪奇孚瑞生物科技有限公司
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