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RT-LAMP detection primer group of spring viremia of carp virus (SVCV), kit and detection method

A technology of RT-LAMP and carp spring viremia, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. Low dimer probability, easy operation, high specific effect

Active Publication Date: 2013-04-17
河北三狮生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It generally takes more than 15 days to obtain the results of cultivating and isolating carp spring viremia virus. The detection cycle is too long to meet the requirements of the era of rapid detection at ports and large customs clearance.

Method used

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  • RT-LAMP detection primer group of spring viremia of carp virus (SVCV), kit and detection method
  • RT-LAMP detection primer group of spring viremia of carp virus (SVCV), kit and detection method
  • RT-LAMP detection primer group of spring viremia of carp virus (SVCV), kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Establishment of carp spring viremia virus RT-LAMP detection kit

[0045] RT-LAMP detection kit for carp spring fish viremia virus, including RT-LAMP primer set, RT-LAMP reaction solution, Bst DNA polymerase, AMV Reverse transcriptase, positive and negative controls.

[0046] (1) RT-LAMP primer design: the envelope glycoprotein gene of carp spring viremia virus (SVCV) ( gly G Gene, GenBank accession number is AY527273.1) as the target gene, using the online design software Primer Explorer version 4 (http: / / primerexplorer.jp / e) to design LAMP primers. The primer sequences are listed in Table 1.

[0047]

[0048] (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.

[0049] (3) The positive control is a T vector clone containing the envelope glycoprotein gene fragment of carp spring viremia virus (SVCV). The outer primers (SEQ ID NO:1 and SEQ ID N...

Embodiment 2

[0051] Example 2 Establishment of carp spring viremia virus RT-LAMP detection method using a turbidimeter:

[0052] The method utilizing the kit of embodiment 1 to detect carp spring carp viremia virus comprises the steps:

[0053] (1) Extraction of RNA from samples to be tested: Extract RNA from samples using a viral RNA extraction kit;

[0054] (2) Constant temperature gene amplification reaction: 25μl reaction system contains: SVCV-F3 0.2μM, SVCV-B3 0.2μM, SVCV-FIP 1.6μM, SVCV-BIP 1.6μM, SVCV-LF 0.8μM, SVCV-LB 0.8μM, RT-LAMP reaction solution 12.5μl, AMV reverse transcriptase 1U, Bst DNA polymerase 8U, RNA to be tested 1~100ng, make up to 25μl with DEPC water; set positive control and negative control; mix the prepared PCR tube and centrifuge, and react at 63~65℃ for 60~90min, and Continue at 80°C for 2 minutes;

[0055] (3) Result judgment: judge the amplification result by observing the turbidity change of the precipitate in the reaction tube. If there is a preci...

Embodiment 3

[0056] Example 3 ESE-Quant tube scanner detection

[0057] The kit is the same as Example 1 except that the chromogenic agent (SYBR Green I) not in Example 1 is added.

[0058] Use the above kit to detect carp spring viraemia virus (SVCV) in the following way:

[0059] (1) Extraction of RNA from the sample to be tested: Extract and purify the RNA from the sample to be tested using a viral RNA extraction kit;

[0060] (2) Constant temperature gene amplification reaction: 25μl reaction system contains: SVCV-F3 0.2μM, SVCV-B3 0.2μM, SVCV-FIP 1.6μM, SVCV-BIP 1.6μM, SVCV-LF 0.8μM, SVCV-LB 0.8μM, RT-LAMP reaction solution 12.5μl, AMV reverse transcriptase 1U, Bst DNA polymerase 8U, 10×SYBR Green I 0.5μl, RNA to be tested 1~100ng, make up to 25μl with DEPC water; set positive control and negative control; mix the prepared PCR tube and centrifuge, react at 65℃ for 60min , and continue at 80°C for 2 minutes;

[0061] (3) Result judgment: place the reaction tube in the ESE-Tube...

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Abstract

The invention discloses an RT-LAMP detection primer group of spring viremia of carp virus (SVCV), a detection kit and a detection method. The detection primer group comprises a pair of outer primers, a pair of inner primers and a pair of cyclic primers; the detection kit comprises primer liquid, reaction liquid, DNA (deoxyribonucleic acid) polymerase, reverse transcriptase and a contrast; and the kit also can be provided with a color developing agent. The detection method comprises the following steps of: extracting the RNA (ribonucleic acid) of the to-be-detected virus; and performing amplification of a sample RNA template at 63-65 DEG C by adopting six specific primers and DNA polymerase with strand displacement activity according to the reverse transcription activity of reverse transcriptase, wherein the pg level of pure virus RNA can be detected, and the identification adds the detection of SYBRGreen IESE-Quant-tubeScanner instrument or the turbidity change of precipitate in a reaction tube is observed by a turbidity meter to judge amplification to determine whether a to-be-detected sample contains the RNA of SVCV. The method disclosed by the invention has the advantages of high speed and high efficiency, convenience in operation, high specificity, high sensitivity, easiness in identification, suitability for field detection and the like, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a detection method for marine aquaculture, in particular to an RT-LAMP detection primer set, a detection kit and a detection method for carp spring viremia virus (SVCV). Background technique [0002] Spring viremia of carp (SVC) is an acute, hemorrhagic infectious disease caused by carp spring viremia virus (SVCV) infecting carps. It is characterized by systemic hemorrhage and ascites, acute onset, and high mortality. It is often prevalent in carps, especially carps, and outbreaks often occur in spring and cause death of juveniles and adults. The disease was first reported in Europe, and then spread widely in carp farming in Europe and caused huge economic losses. It is one of the diseases that need to be reported to OIE according to the International Organization for Animal Health (OIE). In my country, it is also listed as a second-class animal disease in the "List of F...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 李红梅石磊唐大运常彦磊袁忠张璜
Owner 河北三狮生物科技有限公司
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