Novel and practical litopenaeus vannamei RNA (ribonucleic acid) virus fast detection kit and detection method

A technology of RNA virus and detection kit, which is applied in the field of rapid detection kit for Litopenaeus vannamei RNA virus, which can solve the problem of easy contamination of LAMP reaction reagents, false positive results, limitations in the application performance of LAMP detection kits, and unfavorable promotion and application of LAMP reactions and other problems, to achieve the effect of simplifying the steps of RNA extraction, improving operability and simplifying operation

Inactive Publication Date: 2016-07-27
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain deficiencies in the existing technical methods: for example, the detection sensitivity of the cell culture method is not high, and the detection time is long; the PCR detection method requires specific instruments and equipment, and has high requirements for the detection environment; Operation, higher professional requirements for technicians
However, the existing RNA virus LAMP detection kits all need to undergo conventional RNA extraction and purification processes. The RNA extraction method needs to undergo Trizol extraction, chloroform layering, isopropanol extraction and cent

Method used

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  • Novel and practical litopenaeus vannamei RNA (ribonucleic acid) virus fast detection kit and detection method
  • Novel and practical litopenaeus vannamei RNA (ribonucleic acid) virus fast detection kit and detection method
  • Novel and practical litopenaeus vannamei RNA (ribonucleic acid) virus fast detection kit and detection method

Examples

Experimental program
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Example Embodiment

[0036] Example 1:

[0037] (1) Design, synthesis and screening of RT-LAMP detection primer set for TSV virus

[0038] Search for the gene sequence of Litopenaeus vannamei TSV virus in the Genebank database, upload the TSV sequence to the online software PrimerExplorerV4 (http: / / primerexplorer.jp / e / ) to search for LAMP primers, and the 5 sets of LAMP primer sequences obtained, respectively, according to 5'→3' sequence was synthesized, and the total RNA of diseased shrimp containing TSV virus was detected by RT-nested PCR as a template, and 5 sets of primers were used for RT-LAMP reaction. Water and shrimp DNA templates were used as controls to coagulate the reaction products. Gel electrophoresis, screen out the brightest and clearest set of primers as the detection primer set.

[0039] The selected TSV virus LAMP detection primer set is:

[0040] Upstream outer primer F3: 5’-TTCACAGATCATCGACATCT-3’;

[0041] Downstream outer primer B3: 5’-TAGGAGAGACTGTCGGAG-3’;

[0042] Upstream inner p...

Example Embodiment

[0055] Example 2:

[0056] (1) Design, synthesis and screening of RT-LAMP detection primer set for CMNV virus

[0057] Search the gene sequence of Litopenaeus vannamei CMNV virus RNA2 in the Genebank database, upload the gene sequence of CMNVRNA2 to the online software PrimerExplorerV4 (http: / / primerexplorer.jp / e / ) to search for LAMP primers, and then obtain 5 sets of LAMP primer sequences , Were synthesized in the order of 5'→3', and the total RNA of sick shrimp containing CMNV virus detected by RT-nested PCR was used as a template, and 5 sets of primers were used for RT-LAMP reaction. Water and shrimp DNA templates were used as controls to react The product was subjected to gel electrophoresis, and the brightest and clearest set of primers was selected as the detection primer set.

[0058] The RT-LAMP detection primer set of the CMNV virus screened is:

[0059] Upstream outer primer F3: 5’-AGAGCAGAATTTCCAGTCAG-3’;

[0060] Downstream outer primer B3: 5'-ATATTGAAACCTGTGGTTTGT-3';

[0...

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Abstract

The invention discloses a novel and practical litopenaeus vannamei RNA (ribonucleic acid) virus fast detection kit and a detection method. The detection kit comprises a PBS (poly butylenes succinate) pretreatment solution, an RNA fast extraction solution and a detection solution. The kit and the detection method have the advantages that the sensitivity is high; the specificity is high; the detection time is short; the accuracy is high; the operation is simple; the requirements on instruments and equipment are low; the pollution cannot be caused. The problems that the conventional detection method has high requirements on the equipment and the detection environment, the operation is complicated, the requirement on the specialty of technicians is high, and the like are solved. The wide application prospects are realized.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel and practical rapid detection kit and detection method of Litopenaeus vannamei RNA virus. Background technique: [0002] Litopenaeus vannamei (Litopenaeus vannamei, also known as Penaeus vannamei) occupies an absolute dominant position in my country's prawn farming. In 2014, the aquaculture output reached 1.4 million tons, accounting for more than 85% of the country's total prawn aquaculture output. It occupies a relatively large share in China's fishery economy. Large specific gravity. However, disease problems cause huge losses to the shrimp industry every year, seriously restricting the healthy development of the industry. Litopenaeus vannamei RNA virus is one of the more harmful species, such as: Taura virus (TauraSyndromeVirus, TSV) can cause 60-90% of catastrophic mortality of Litopenaeus vannamei juveniles, stealing dead disease Nomura virus (Covertmortali...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 黄文任春华罗鹏赵哲江晓胡超群陈廷王艳红
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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