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Primer set for performing fluorescence quantitative PCR detection on decapod iridescent virus 1 ( Decapod iridescent virus 1, DIV1 ) and reagent kit

A detection kit and fluorescence quantitative technology, which are applied in the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of false positive, cumbersome operation, easy cross-contamination, etc. High sensitivity and good repeatability

Pending Publication Date: 2020-06-02
GUANGXI ACADEMY OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, at present, there are no relevant standards for DIV1 detection, both internationally and domestically, and the Ministry of Agriculture and Rural Affairs recommends the nested PCR method for detection in the DIV1 monitoring plan; Two rounds of PCR and electrophoresis are required, the operation is cumbersome, the detection time is long, and the cover is easily cross-contaminated, especially in laboratories with poor PCR partition conditions, it is easy to form aerosols to pollute the environment and cause false positives
[0005] In addition, the rapid detection methods of DIV1 include TaqMan probe quantitative PCR method and loop-mediated constant temperature amplification technique (LAMP), but the sensitivity of TaqMan probe quantitative PCR is not ideal because the probe length is too long; LAMP is sensitive and fast, but Prone to false positives under laboratory conditions

Method used

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  • Primer set for performing fluorescence quantitative PCR detection on decapod iridescent virus 1 ( Decapod iridescent virus 1, DIV1 ) and reagent kit
  • Primer set for performing fluorescence quantitative PCR detection on decapod iridescent virus 1 ( Decapod iridescent virus 1, DIV1 ) and reagent kit
  • Primer set for performing fluorescence quantitative PCR detection on decapod iridescent virus 1 ( Decapod iridescent virus 1, DIV1 ) and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Design and synthesis of primers and TaqMan-MGB probe for detecting Decapod iridescent virus 1 (DIV1).

[0043] The major capsid protein (MCP) gene of iridovirus contains many highly conserved domains, so the MCP gene of DIV1 was used as the detection target gene. Using Primer Express 3.0 software, according to the design principles of fluorescent quantitative PCR primers and MGB probes, multiple sets of specific amplification primers and TaqMan-MGB probes were designed and screened in the conserved region of the MCP gene (KY681039), and analyzed and compared by Oligo7.0 software. Appropriately modify and optimize to obtain 5 groups of ideal primers and probes (see Table 1). After Blast homology comparison and verification through the NCBI database, they will be synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd., wherein the 5' end of the probe Label the fluorescent dye FAM, and label the MGB group at the 3' end.

[0044] Table 1 Primer and probe information f...

Embodiment 2

[0051] Extraction of pathogenic nucleic acid and preparation of recombinant plasmid standards.

[0052] Take 50mg of positive diseased tissue, and follow the instructions of the animal tissue genomic DNA rapid extraction kit to extract common shrimp pathogens DIV1, WSSV, IHHNV, EHP and Vp AHPND Finally, add 50 μL of elution buffer (EB) to dissolve the DNA, and store at –20°C for later use.

[0053] Using the DNA extracted from the DIV1-positive material as a template, PCR amplification was performed using primers DIV1-qF1 and DIV1-qR1 (amplification conditions are the same as in Example 1), and the product was recovered and purified by agarose gel and ligated with pMD18-T to transform DH5α , to prepare the recombinant plasmid pMD18-T-MCP DIV1 , and identified by PCR and sequencing. Extract the recombinant plasmid pMD18-T-MCP DIV1 , use a nucleic acid and protein analyzer to measure its concentration and convert it to copy number, and dilute to 1.0×10 in a 10-fold gradient ...

Embodiment 3

[0056] Optimization of reaction conditions for fluorescent quantitative PCR.

[0057] With 3 gradients (1.0×10 5 , 1.0×10 3 , 1.0×10 1 copies / μL) of the standard DNA as a template, in the range of 56 ~ 64 ° C with a gradient of 1 ° C as the annealing temperature for fluorescent quantitative PCR amplification (other conditions are the same as in Example 1), to obtain a lower threshold cycle number (Ct value) and higher relative fluorescence intensity increase value (ΔRn) is the best annealing temperature.

[0058] The results show that when the annealing temperature is 58°C, the Ct value is the smallest and the ΔRn is the highest, indicating that both the primer and the probe can be well combined with the template when the annealing temperature is 58°C, so 58°C is selected as the optimal annealing temperature in the present invention. Among them, the initial template concentration was 1.0 × 10 3 Refer to Table 3 for the amplification effect data of the fluorescent quantitat...

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Abstract

The invention discloses a primer set for performing fluorescence quantitative PCR detection on decapod iridescent virus 1 ( Decapod iridescent virus 1, DIV1 ) and a reagent kit, and belongs to the technical field of virus detection. The primer set specifically comprises a primer for detecting the decapod iridescent virus 1 and a TaqMan-MGB probe, and a reagent kit based on the detection primer andthe probe. Through the primer set disclosed by the invention, according to an MCP gene consensus sequence of DIV1, a specific primer and the TaqMan-MGB probe are designed, a recombinant plasmid standard product pMD18-T-MCPDIV1 is prepared, a TaqMan-MGB probe fluorescence quantitative PCR method for detecting DIV1 is established, the TaqMan-MGB probe fluorescence quantitative PCR method has the advantages of being high in sensitivity, high in specificity, good in repeatability, wide in quantitative range, simple and quick and the like; and a corresponding detection reagent kit is researched and developed, and popularization and application are convenient, so that quick quantitative detection of the DIV1 and the prevention and control of relevant diseases are facilitated.

Description

technical field [0001] The invention relates to the technical field of virus detection, and more specifically relates to a primer set and a kit for detecting decapod iridescent virus 1 by fluorescent quantitative PCR. Background technique [0002] Decapod iridescent virus 1 (DIV1) is a new type of iridescent virus newly discovered and identified in recent years. DIV1 can infect various economic farming species such as Litopenaeus vannamei, Macrobrachium rosenbergii, crayfish, and Penaeus chinensis. Shrimp can also cause large-scale deaths, and can also infect common aquatic organisms such as plankton, river shrimp, crucian carp, snails, and river crabs to become the source of infection and spread the virus; DIV1 has a variety of susceptible hosts, making it easier to spread and become popular. It has caused a large number of deaths of aquaculture shrimp outbreaks in many regions of my country, and has become a new threat to China's shrimp aquaculture industry. [0003] At pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2545/114Y02A50/30
Inventor 童桂香韦信贤林勇谭红连杨慧赞黄光华杨琼王瑞李旻熊建华
Owner GUANGXI ACADEMY OF FISHERY SCI
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