Siniperca chuatsi ranairidovirus and rhabdovirus duplex PCR detection kit and detection method

A rhabdovirus and frog virus technology, applied in the field of PCR, can solve time-consuming and labor-intensive problems, and achieve accurate detection and high sensitivity

Pending Publication Date: 2020-06-09
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, epidemiological studies have found that there is a phenomenon of mixed infection of SCRIV and SCRV. At present, laboratory tests are mainly carried out by PCR for SCRIV and SCRV respectively. Two PCRs are required to detect different viruses. This method is time-consuming and laborious.

Method used

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  • Siniperca chuatsi ranairidovirus and rhabdovirus duplex PCR detection kit and detection method
  • Siniperca chuatsi ranairidovirus and rhabdovirus duplex PCR detection kit and detection method
  • Siniperca chuatsi ranairidovirus and rhabdovirus duplex PCR detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] This example is to construct the standard recombinant plasmids P-SCRIV and P-SCRV as the positive control solution of the kit of the present invention.

[0060] experiment method:

[0061] 1. Use the DNA of clinical samples infected with SCRIV as a template and use SCRIV-MCP-F / SCRIV-MCP-R as primers for PCR amplification. The total PCR reaction system is 25 μL: 12.5 μL of 2×EasyTaq PCR SuperMix, 1 μL of primers SCRIV-MCP-F / SCRIV-MCP-R, 1 μL of template, and sterile ddH 2 O supplemented to 25 μL. At the same time ddH2O was used instead of the template as a negative control. The parameters of the PCR amplification reaction were: pre-denaturation at 94°C for 5 min; 30 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min and 30 s; final extension at 72°C for 10 min. The reaction products were detected by 1% agarose gel electrophoresis. After the PCR amplification product is purified, take 4 μL and connect it with 1 μL pEASY-T1 vector, and transfer it into Escheric...

Embodiment 2

[0068] Present embodiment is a kind of mandarin fish frog virus (SCRIV) described in the present invention and mandarin fish frog virus (SCRV) double PCR detection kit and establishes the double PCR detection method of SCRIV and SCRV

[0069] 1. A double PCR detection kit for mandarin fish frog virus (SCRIV) and mandarin fish rhizome virus (SCRV), comprising adding 1 μL, 2× EasyTaq PCR SuperMix 12.5 μL, positive control solution 2 μL, and negative control solution to complement each primer respectively. 25 μL; wherein, the primers include primers SCRIV-400-F / SCRIV-400-R designed for the conserved region of the MCP gene of mandarin fish frog virus, and primers designed for the conserved region of the N gene of mandarin fish rhabdovirus SCRV-280-F / SCRV-280-R, its primer sequence is shown in Table 1; The positive control solution is the mixture P-SCRIV 1 μL+P-SCRV 1 μL of the standard recombinant plasmid constructed in Example 1, the negative control ddH 2 O.

[0070] 2. Establ...

Embodiment 3

[0083] This embodiment is the optimization of the amplification conditions of SCRIV and SCRV double PCR detection method

[0084] experiment method:

[0085] In this embodiment, the SCRIV and SCRV double PCR detection methods are the same as in Embodiment 2, except that the annealing temperature and the amount of primers are different. The annealing temperature is sequentially changed from 57°C, 58°C, 59...66°C, and detected by 2% agarose gel electrophoresis. After determining the optimal annealing temperature, optimize the amount of primers, (SCRIV-400-F / SCRIV-400-R) or (SCRV-280-F / SCRV-280-R) primer amount was set to 0.5 / 0.5 ( The upstream and downstream primers of each pair of primers are the same amount), 0.5 / 1, 0.5 / 1.5, 1 / 0.5, 1 / 1, 1 / 1.5, 1.5 / 0.5, 1.5 / 1 and 1.5 / 1.5. ddH 2 O was used as a negative control instead of the template, and the PCR amplification product obtained was detected by electrophoresis on 2% agarose gel.

[0086] test results:

[0087] Optimal anneal...

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Abstract

The invention relates to a siniperca chuatsi ranairidovirus and rhabdovirus duplex PCR detection kit and detection method, and belongs to the technical field of PCR. The siniperca chuatsi ranairidovirus and rhabdovirus duplex PCR detection kit provided by the invention comprises primers, EasyTaq PCR SuperMix, a negative control solution and a positive control solution, wherein the primers comprisea primer designed by aiming at an MCP gene conserved region of the siniperca chuatsi ranairidovirus, and a primer designed by aiming at an N gene conserved region of the siniperca chuatsi rhabdovirus. The siniperca chuatsi ranairidovirus and rhabdovirus duplex PCR detection kit and detection method provided by the invention can be used for fast, efficiently and accurately detecting the sinipercachuatsi ranairidovirus and the siniperca chuatsi rhabdovirus at the same time; the time and the labor are saved; more treatment time is won for sick siniperca chuatsi; and important significance is realized on subsequent study, prevention and control.

Description

technical field [0001] The invention relates to a double PCR detection kit and detection method of mandarin fish frog virus and rhabdovirus, belonging to the technical field of PCR. Background technique [0002] Mandarin fish is a species favored by aquaculture farmers in recent years. Its meat is delicious and has high nutritional value, and it is especially popular with the masses. However, Siniperca chuatsi ranairidovirus (SCRIV) and Siniperca chuatsi rhabdovirus (SCRV) are the main viral pathogens causing mandarin fish disease in recent years, and they are the two most harmful to the mandarin fish aquaculture industry. Once infected with the virus, the survival rate of mandarin fish is extremely low, and there is no effective treatment method, which brings great economic losses to farmers. [0003] Currently, the most common method for detecting SCRIV and SCRV viruses in the laboratory is the polymerase chain reaction assay. Polymerase Chain Reaction (Polymerase Chain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 梁红茹黄瑜聪李宁求林强付小哲刘礼辉牛银杰黄志斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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