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LAMP (Loop-mediated Isothermal Amplification) detection primer group, kit and method for shrimp blood cell iridovirus

A detection kit and technology for iridescent virus, applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as easy missed detection, achieve strong specificity, improve the correct detection rate, and high sensitivity Effect

Pending Publication Date: 2021-05-14
珠海市迪奇孚瑞生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Among them, the invention patent application with publication number CN108950067A discloses a LAMP primer set, kit and method for detecting prawn iridescent virus. The LAMP primer set includes a pair of outer primers, a pair of inner primers and a pair of loop primers. Although the detection kit can specifically amplify the shrimp iridescent virus, there are the following technical problems: when the virus DNA content in the early stage of infection is lower than the detection limit of the sample, it is easy to miss the detection

Method used

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  • LAMP (Loop-mediated Isothermal Amplification) detection primer group, kit and method for shrimp blood cell iridovirus
  • LAMP (Loop-mediated Isothermal Amplification) detection primer group, kit and method for shrimp blood cell iridovirus
  • LAMP (Loop-mediated Isothermal Amplification) detection primer group, kit and method for shrimp blood cell iridovirus

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1, the design of the detection primer group of shrimp hemocyte iridescent virus LAMP

[0038] The present invention designs a LAMP detection primer set by comparing the differences of the shrimp hemocyte iridescent virus. The LAMP detection primer set is not only used for specific amplification of the shrimp hemocyte iridescent virus DNA, but also for the specific reversal of the RNA produced by the shrimp hemocyte iridescent virus. Recorded, the LAMP detection primer set includes six specific primers OF, OR, IF, IR, LF, LR, the specific sequences are as follows:

[0039] OF: 5'-AATGTTGGGAAAGTTTGCA-3';

[0040] OR: 5'-CCTTTCCTCGTTGGAACAA-3';

[0041] IF: 5'-CATCTAACACCATCTCCCGCCTCTGATTACGGGTAAAAAAGGC-3';

[0042] IR: 5'-AGTCATGGATGAACCAAATGCTGACGAGCCCAATACGAATCG-3';

[0043] LF: 5'-CCAATTCGGGACTTGCAG-3';

[0044] LR: 5'-GAACGTTAAAGGGTCTCACG-3'.

[0045]By designing six specific primers OF, OR, IF, IR, LF, and LR, the present invention can not only specif...

Embodiment 2

[0046] Example 2, Establishment of Shrimp Hemocyte Iridescent Virus RT-LAMP Detection Kit

[0047] The shrimp hemocyte iridovirus RT-LAMP detection kit includes the shrimp hemocyte iridovirus LAMP detection primer set in Example 1, a LAMP reaction solution, a color reagent, a positive control group and a negative control group.

[0048] The LAMP reaction solution includes 5× constant temperature amplification buffer, dNTP, DNA polymerase and reverse transcriptase; the DNA polymerase is Bst DNA polymerase; the reverse transcriptase is HISCRIPTIII reverse transcriptase; the chromogenic reagent includes SYBRTMGreenI fluorescent dye.

[0049] The positive control group is the T vector containing the ATPase gene fragment of shrimp hematocytic iridescent virus.

[0050] The negative control group was ultrapure water.

Embodiment 3

[0051] Example 3, Establishment of RT-LAMP Detection Method for Shrimp Hemocyte Iridescent Virus

[0052] 1. Extraction of nucleic acid from samples to be tested

[0053] The sample to be tested in the embodiment of the present invention is shrimp fry or adult shrimp tissue, and the nucleic acid (including DNA and RNA) of the sample to be tested is extracted by grinding, and the operation is as follows:

[0054] 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;

[0055] 1.2 Add 500ul lysate, mix thoroughly; lyse at room temperature for 10 minutes;

[0056] 1.3 Use a centrifuge to centrifuge at 12000g for 1 minute, take the supernatant, and add it to the nucleic acid adsorption column;

[0057] 1.4 Use a centrifuge with a centrifugal force of 8000g for centrifugation, so that the supernatant passes through the adsorption column, ...

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Abstract

The invention discloses a shrimp blood cell iridovirus LAMP (loop-mediated isothermal amplification) detection primer group, a kit and a method, the primer group comprises a primer OF, a primer OR, a primer IF, a primer IR, a primer LF and a primer LR, oR is 5 '-CCTTTCCTCGTTGGAACA-3', and OR is 5 '-CCTTTCCTGTTGGAACA-3 iF: 5 '-CATCTAACACCATCTTCCCGCCTCTGATTACGGGTAAAAAGGC-3', and IF: 5 '- in the formula, IR is 5 '-AGTCATGGATGAACCAATGCTGACGAGCCCAATACGAATCG-3', and R is 5 '- the LF is 5 '-CCAATTCGGGACTTGCAG-3', the LF is 5 '- the LR is 5 '-GAACGTTAAAGGGTTCACCG-3', and the LR is 5 '- according to the method, not only can the DNA of the prawn blood cell iridovirus be subjected to specific amplification, but also the RNA generated by the prawn blood cell iridovirus can be subjected to specific reverse transcription, so that the problem that in the prior art, the virus DNA content in the initial stage of infection of the prawn blood cell iridovirus is often lower than the detection limit of a detection product, so that missing detection occurs is solved; by simultaneously detecting the DNA of the shrimp blood cell iridovirus and the RNA which is generated by mass transcription of the shrimp blood cell iridovirus and has the same sequence as the DNA, the correct detection rate of the shrimp blood cell iridovirus is improved, the detection time is shortened, and the specificity and the sensitivity are high.

Description

technical field [0001] The invention relates to the technical field of biological virus detection, in particular to a primer set, kit and detection method for shrimp hemocyte iridescent virus LAMP detection. Background technique [0002] In recent years, in East Asia, Southeast Asia and Europe, fish diseases caused by iridescent viruses have caused major economic losses to the aquaculture industry, seriously hindering the healthy development of the fish farming industry, and have received more and more attention at home and abroad . The family Iridoviridae is divided into five virus genera, namely, Iridovirus, Chloriridovirus, Lymphocystivirus, Ranavirus, and cytomegalovirus (Megalocytivirus), mainly infects invertebrates and lower vertebrates. In 2017, researchers from the Yellow Sea Research Institute of the Chinese Academy of Sciences isolated and identified a new virus from a batch of severely ill and dead Litopenaeus vannamei samples in Zhejiang Province. After histop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2521/107C12Q2563/107
Inventor 陈天蓝罗达圣董铖唐翔
Owner 珠海市迪奇孚瑞生物科技有限公司
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