RAA amplification primer and probe for rapidly detecting carp edema virus, detection kit and using method

A technique for amplifying primers and kits, applied in biochemical equipment and methods, methods based on microorganisms, DNA/RNA fragments, etc. Equipment and other problems, to achieve rapid response, sensitive detection, and high specificity

Inactive Publication Date: 2019-09-06
北京市水产技术推广站
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods require expensive instruments and equipment, high testing costs, and high technical requirements for testing personnel, and are difficult to meet non-laboratory on-site rapid testing and grassroots popularization and application.

Method used

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  • RAA amplification primer and probe for rapidly detecting carp edema virus, detection kit and using method
  • RAA amplification primer and probe for rapidly detecting carp edema virus, detection kit and using method
  • RAA amplification primer and probe for rapidly detecting carp edema virus, detection kit and using method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The establishment of the RAA-LFD rapid detection kit of embodiment 1 carp edema virus

[0036] 1. Design and synthesis of RAA primers and probes for carp edema virus

[0037] Taking the conserved region of the CEV P4a gene in the NCBI database as the target site, according to the principle of RAA primer design, DNAman 6.0 software was used for sequence alignment analysis, and fragments with high homology were selected, and 4 pairs of primers were designed with primer primer 5.0 (Table 1). 1).

[0038] Primers used in Table 1 RAA:

[0039]

[0040] 2. CEV RAA Detection Primer Screening

[0041] With CEV positive plasmid (6.14×10 6 copies / μL) as the template for amplification, after the reaction, use 2% agarose electrophoresis to identify (see figure 1 ), screen the designed primers, and screen out the optimal primer pair as the second set of primers, and the optimal primer pair is F and R:

[0042] Upstream primers:

[0043] CEV-2-F: 5'-AGATTGTAGCATTTCCTAGTTTGTA...

Embodiment 2

[0055] Embodiment 2 Utilizes the method for detecting carp edema virus RAA-LFD rapid detection kit described in embodiment 1

[0056] 1. Tissue pretreatment and DNA extraction: Take 30-80 mg of gill and kidney tissues of carp or koi carp, homogenize at low temperature, add PBS buffer or M199 cell culture medium at a ratio of 1:10, freeze and thaw once, 1000r / After centrifugation for 10 min, take 200 μL of the supernatant, and use the traditional phenol-chloroform reagent or an equivalent DNA extraction kit for DNA extraction.

[0057] 2. The configuration of the carp edema virus RAA reaction system: each test sample corresponds to a RAA reaction dry powder tube, and the reaction components and the added volume in each RAA reaction dry powder tube are shown in Table 2.

[0058] Table 2:

[0059] RAA reaction system components Volume (μL) A Buffer 40.9 primer mix 4 specific probe 0.6 DNA template 2 B Buffer 2.5 total capacity 50 ...

Embodiment 3

[0064] The sensitivity test of embodiment 3 kits of the present invention

[0065] The carp edema virus positive plasmid standard product that kit described in the embodiment of the present invention 1 provides, measures concentration with NanoDrop, according to formula﹛plasmid concentration (ng / μL) * 10 -9 / [660×(plasmid length+vector length)]﹜×6.023×10 23 = Plasmid copy number (copies / μL) converted to its copy number. The plasmids were divided into 6.14×10 8 to 6.14×10 0 Copy / μL was diluted in 9 concentration gradients for sensitivity test.

[0066] Test results such as figure 2 As shown, except the negative control, from left to right is 6.14×10 0 ~6.14×10 8 The amplification result of the positive standard product of copy / μL, can find out that the RAA-LFD sensitivity minimum detection limit of the present invention is 6.14 * 10 0 copy / μL, the sensitivity is better than that of ordinary PCR detection method, and is equivalent to fluorescent PCR, indicating that the ...

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Abstract

The invention discloses a RAA amplification primer and probe for rapidly detecting carp edema virus, a detection kit and a using method. The RAA isothermal amplification system is fast in reaction andwide in temperature range, effective amplification of target genes can be realized at the temperature of 34-40 DEG C, the minimum detection limit of the RAA isothermal amplification system is 6.14* 10 degrees copies / mu L, the sensitivity is equivalent to that of TaqMan fluorescence quantitative PCR of CEV, and the RAA isothermal amplification system is not in cross reaction with other aquatic animal epidemic diseases. The kit provided by the invention can quickly, efficiently and sensitively detect the carp edema virus, has the characteristics of simplicity in operation, high specificity, easiness in observation of reaction results, safety, no pollution and the like, not only provides an effective technical means for on-site rapid detection and screening of nucleic acid infected by the carp edema virus, but also has quite important significance for controlling the infection transmission of the carp edema virus in Chinese carps and koi carps, and inspection and quarantine of the carp edema virus in epidemic areas and entry-exit ports.

Description

technical field [0001] The invention relates to the field of aquatic animal diseases in the aquaculture industry, in particular to a RAA amplification primer and probe for rapid detection of carp edema virus, a detection kit and a use method. Background technique [0002] Carp edema virus disease (CEVD), also known as koi sleeping sickness (koi sleepydisease, KSD), is a highly contagious disease caused by a poxvirus carp edema virus (Carp edema virus, CEV) infecting carp and koi. epidemic. Diseased fish often have symptoms such as gill rot, sunken eyes, and lethargy. The mortality rate caused by the disease is as high as 80-100%, causing serious economic losses to the aquaculture industry. The disease has spread rapidly around the world, and CEVD was first reported in my country in 2016, which is a new epidemic of aquatic animals in my country. [0003] At present, there is no effective treatment for carp edema caused by CEV, and prevention is the main method. Therefore, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/94
CPCC12Q1/701C12Q1/6844
Inventor 吕晓楠徐立蒲张文曹欢王小亮王姝王静波
Owner 北京市水产技术推广站
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