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Triple fluorescent PCR detection kit for infectious spleen and kidney necrosis virus, largemouth bass ranavirus and siniperca chuatsi rhabdovirus

A spleen-kidney necrosis virus and kit technology, applied in the field of triple fluorescent PCR detection kits, can solve the problems of economic loss in fish farms, difficulty in judging virus infection, and difficulty in identifying pathogens in advance, achieving good application prospects and specificity Good, high-sensitivity effect

Active Publication Date: 2020-10-02
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0005] Infectious spleen and kidney necrosis virus (ISKNV), largemouth bass virus (LMBV), and mandarin fish fluke virus (SCRV) are three common and easily infectious pathogens in freshwater farmed fish, each of which can cause large-scale outbreaks and popular, causing serious economic losses
[0006] Since freshwater fish are now generally farmed on a large scale, and there is no vaccine or treatment for the three viruses, if the virus infection cannot be found in the seedling stage, or early in the breeding process, and take corresponding prevention and control measures Measures will cause huge economic losses to fish farms; the three viruses all have similar bleeding symptoms during the onset period, and it is difficult to judge the virus infection from the appearance; and in the early stage of virus infection, when there are no obvious clinical symptoms, It is difficult to identify pathogens in advance by traditional observation method or ordinary PCR method

Method used

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  • Triple fluorescent PCR detection kit for infectious spleen and kidney necrosis virus, largemouth bass ranavirus and siniperca chuatsi rhabdovirus
  • Triple fluorescent PCR detection kit for infectious spleen and kidney necrosis virus, largemouth bass ranavirus and siniperca chuatsi rhabdovirus
  • Triple fluorescent PCR detection kit for infectious spleen and kidney necrosis virus, largemouth bass ranavirus and siniperca chuatsi rhabdovirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1 Primer and probe design

[0074] The present inventor designed 3 proteins according to the Infectious Spleen and Kidney Necrosis Virus (ISKNV) MCP protein sequence DQ399789 registered in Genebank, the mandarin fish flutter virus (SCRV) nucleocapsid protein sequence DQ399789, and the largemouth bass virus (LMBV) MCP protein sequence JQ178325. Article-specific probes and 3 pairs of primers (Table 1), including: ISKNV-rtF (SEQ ID NO:1), ISKNV-rtR (SEQ ID NO:2), ISKNV-rtProbe (SEQ ID NO:3), SCRV -rtF (SEQ ID NO:4), SCRV-rtR (SEQ ID NO:5), SCRV-rtProbe (SEQ ID NO:6), LMBV-rtF (SEQ ID NO:7), LMBV-rtR (SEQ ID NO :8) and LMBV-rtProbe (SEQ ID NO:9); the probe has been verified by Blast and has high specificity. Synthesized by Invitrogen Company.

[0075] Table 1 Triple fluorescent PCR probes and amplification primers

[0076]

Embodiment 2

[0077] The preparation of embodiment 2 nucleic acid sample and standard

[0078] Refer to the instructions of the viral DNA / RNA mini-extraction kit to extract the total nucleic acid from the diseased tissues of mandarin fish and perch as the amplification template, and store it at -20°C for later use. Using ISKNV, LMBV and SCRV whole viral genome nucleic acids as templates respectively, ISKNV-F / R (SEQ ID NO: 10-11), SCRV-F / R (SEQ ID NO: 12-13), LMBV-F / R (SEQ ID NO:14~15) (used concentration: 10μM) was used as primers for PCR or RT-PCR amplification respectively, and the DNA virus amplification conditions were: 94°C for 3min; 94°C for 30s, 55°C for 30s, 72°C for 45s , a total of 35 cycles; 72°C for 10 minutes; RNA virus amplification conditions were 50°C, 15 minutes, 94°C for 3 minutes; 94°C for 30s, 55°C for 30s, 72°C for 45s, a total of 35 cycles; 72°C for 10 minutes. The reaction products were detected and separated by 2% agarose gel electrophoresis. After the PCR products...

Embodiment 3 3

[0081] Example 3 Establishment of Triple Fluorescent PCR Freeze-drying System and Optimization of Reaction Conditions

[0082] The positive standard substance that embodiment 2 prepares is diluted to 10 with nuclease-free water respectively 4 、10 3 、10 2 , 10 1 、10 0 copies / μL, then the same concentration of positive standard dilutions were mixed in equal proportions, and then this mixture was used as a template for sensitivity optimization; the total nucleic acid of the extracted mandarin rhizoma virus (SCRV) positive disease material was used as reverse transcriptase and RNase inhibitor optimized template.

[0083] Prepare a triple fluorescent PCR freeze-dried system, including Taq enzyme, reverse transcriptase, RNase inhibitor, reaction buffer, dNTP, primers and probes corresponding to the three viruses (as shown in Table 1), Nuclease-free water and lyoprotectant. Wherein the lyoprotectant is selected from at least one of trehalose, raffinose, mannitol and glycine.

...

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Abstract

The invention belongs to the technical field of virus detection, and specifically relates to a triple fluorescent PCR detection kit for the infectious spleen and kidney necrosis virus, largemouth bassranavirus and siniperca chuatsi rhabdovirus. The kit of the invention comprises virus-specific amplification primers and probes, a positive standard, a negative standard, Taq enzyme, reverse transcriptase, a RNA enzyme inhibitor, a reaction buffer, dNTP, nuclease-free water and a freeze-drying protective agent. The kit of the invention can perform multiple channel detection at the same time by using triple fluorescent PCR, uses the different fluorescently labeled probes, can detect the three viruses simultaneously in one reaction system, reduces the detection difficulty, shortens the detection time, and can help raisers accurately get detection results in time. The kit of the invention has high detection sensitivity, high stability and strong specificity, has no cross-reactions with otheraquatic viruses, and can be used for early monitoring and prevention and control of epidemic diseases.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a triple fluorescent PCR detection kit for infectious spleen and kidney necrosis virus, largemouth bass virus and mandarin fish flume virus. Background technique [0002] Infectious spleen and kidney necrosis is also known as the explosive infectious disease of mandarin fish. In 1994, an infectious disease with high mortality broke out in the mandarin fish farming area of ​​Guangdong; the main pathogen was extracted from the sick mandarin fish and named as Infectious spleen and kidney necrosis virus (ISKNV) , mainly infecting the spleen and kidney, manifested as enlargement of the diseased organs, and the mortality rate of mandarin fish within 7 to 12 days after infection can reach 100%; in addition, it can also infect more than 50 kinds of sea and freshwater cultured fish. ISKNV belongs to the genus of iridovirus and cytomegalovirus; the pathogenic gene is d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2563/107C12Q2537/143
Inventor 张钰薇伍建敏李中圣曹梦蕊罗律王凤求
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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