RT-LAMP test kit for viral hemorrhagic septicemia virus
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A technology of RT-LAMP and viral hemorrhage, applied in the field of molecular biology, can solve the problems of long detection period, unsuitable for rapid detection, etc., and achieve the effects of simple operation, mild conditions and high sensitivity
Inactive Publication Date: 2014-11-26
东莞出入境检验检疫局检验检疫综合技术中心
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The culture and isolation of viral hemorrhagic sepsis virus usually takes more than 15 days to get the results, and the detection cycle is too long to meet the requirements of the era of rapid detection at ports and large customs clearance
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Embodiment 1
[0041] Establishment of RT-LAMP detection kit for viral hemorrhagic sepsis virus
[0042] RT-LAMP detection kit for viral hemorrhagic sepsis virus, including RT-LAMP primer set, RT-LAMP reaction solution, Bst DNA polymerase, AMV reverse transcriptase, positive control and negative control.
[0043](1), RT-LAMP primer design: with the envelope glycoprotein gene (gly G gene, GenBank accession number DQ401190.1) of viral hemorrhagic sepsis virus (VHSV) as the target gene, use the online design software Primer Explorer version4( http: / / primerexplorer.jp / e) to design LAMP primers. The primer sequences are listed in Table 1.
[0044] Table 1 Primer sequence list
[0045]
[0046] (2) RT-LAMP reaction solution: containing 10mM dNTP, 10×ThermoPol reaction buffer, and 150mM MgSO4 aqueous solution, the volume ratio of the three is 8:5:2.
[0047] (3), positive control is the T vector clone that contains viral hemorrhagic septicemia virus (VHSV) envelope glycoprotein gene fragment,...
Embodiment 2
[0054] ESE-Quant tube scanner detection
[0055] Except that the chromogen (SYBR Green I) not in Example 1 is added in the kit, all the others are the same as Example 1.
[0056] Detect viral hemorrhagic septicemia virus (VHSV) by the following method with above-mentioned kit:
[0057] (1) Extraction of the RNA of the sample to be tested: the RNA of the sample to be tested is extracted and purified using a viral RNA extraction kit;
[0058] (2) Constant temperature gene amplification reaction: 25μl reaction system contains: VHSV-F3 0.2μM, VHSV-B30.2μM, VHSV-FIP 1.6μM, VHSV-BIP 1.6μM, VHSV-LF 0.8μM, VHSV-LB 0.8μM , 12.5 μl of RT-LAMP reaction solution, 1U of AMV reverse transcriptase, 8U of Bst DNA polymerase, 0.5 μl of 10×SYBR Green I, 1 to 100 ng of RNA to be tested, filled up to 25 μl with DEPC water; set positive and negative controls; Mix the prepared PCR tube and centrifuge, react at 65°C for 60 minutes, and continue at 80°C for 2 minutes;
[0059] (3) Judgment of resu...
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Abstract
The present invention belongs to the technical field of molecular biology, and relates to aquatic marine aquaculture industry detection method, particularly to a RT-LAMP test primer set, a test kit and a test method of viral hemorrhagic septicemia virus. The test primer set comprises outer primers, inner primers and ring primers. The test kit mainly comprises primer liquids, reaction solutions, DNA polymerase, reverse transcriptase and controls. The test method comprises that: RNA of virus to be tested is extracted, reverse transcription activity of the reverse transcriptase is utilized, and six specific primers and DNA polymerase with strand displacement activity are adopted to amplify the sample RNA template, wherein the pg grade pure viral RNA can be tested; and identification adopts SYBR Green I addition and ESE-Quant-tube Scanner instrument detection or adopts a turbidity instrument to determine whether the sample contains the viral hemorrhagic septicemia virus RNA. According to the present invention, advantages of rapidness, high efficiency, simple operation, high specificity, high sensitivity, simple identification, easy on-site test, and the like are provided, and popularization and application are easily achieved.
Description
technical field [0001] The invention belongs to the technical field of molecular biology, relates to a detection method for marine aquaculture industry, in particular to an RT-LAMP detection primer set, a detection kit and a detection method for viral hemorrhagic sepsis virus (VHSV). Background technique [0002] Viral hemorrhagic septicemia (VHS) is a disease that infects salmon and trout of all ages and a variety of free-living marine fish (such as cod, herring, sole, catfish, northern pike and turbot, flounder, etc.) Fatal, systemic infectious disease. It is usually popular at the end of winter and early spring and when the water temperature is 8-10°C. When the water temperature rises above 15°C, the incidence rate decreases. The disease is endemic throughout continental Europe, North America, and Japan, and is highly contagious, with 1,000 viruses per liter of water in affected fish farms. The main feature of viral hemorrhagic sepsis is bleeding, and the incubation per...
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