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RAA amplification primer and probe for rapid detection of Cyprinid herpesvirus type II, detection kit and application method thereof

A technology of carp herpes virus and amplification primers, which is applied in the field of RAA amplification primers, probes and detection kits for rapid detection of carp herpes virus type II, and can solve the problems of immunohistochemical methods that need to be verified and virus cell lines that need to be improved, etc. problem, to achieve the effect of rapid response, high specificity and simple operation

Pending Publication Date: 2020-04-07
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

However, these methods have certain limitations: cell lines that can isolate viruses need to be improved, and immunohistochemical methods based on monoclonal antibodies need to be verified

Method used

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  • RAA amplification primer and probe for rapid detection of Cyprinid herpesvirus type II, detection kit and application method thereof
  • RAA amplification primer and probe for rapid detection of Cyprinid herpesvirus type II, detection kit and application method thereof
  • RAA amplification primer and probe for rapid detection of Cyprinid herpesvirus type II, detection kit and application method thereof

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Experimental program
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Effect test

Embodiment 1

[0035] The establishment of the RAA-LFD rapid detection kit of embodiment 1CyHV-2

[0036] 1. Design and synthesis of RAA primers and probes for CyHV-2

[0037] Taking the conserved region of the helicase gene of CyHV-2 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 (as in Table 1).

[0038] Table 1 Primers used in RAA

[0039]

[0040] 2. CyHV-2RAA detection primer screening

[0041] With CyHV-2 positive plasmid (8.14×10 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 third group of primers, and the optimal primer pair is F and R:

[0042] Upstream primers:

[0043] RAA-GFHNV-3-F: 5'-ATCTTCAAGCCCAAGATCAATC...

Embodiment 3

[0063] The sensitivity test of embodiment 3 kits of the present invention

[0064] The CyHV-2 positive plasmid standard substance 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 8.14×10 8 to 8.14×10 0 Copy / μL 9 concentration gradient dilutions were used for sensitivity test.

[0065] Test results such as figure 2 As shown, except the negative control, from left to right is 8.14×10 0 ~8.14×10 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 8.14 * 10 0 copy / μL, the sensitivity is better than that of ordinary PCR detection method, which shows that the RAA-LFD rapid detection kit and detection me...

Embodiment 4

[0066] The specificity test of embodiment 4 kits of the present invention

[0067] In order to detect the specificity of the kit of the present invention, the detection method in Example 2 is used to detect the positive samples of viruses CyHV-2, IHNV, SVCV, GV, CEV, KHV, EHNV, CCV respectively, and analyze the effect of the kit on CyHV -2 and other common DNA viruses in aquatic animals.

[0068] The test results showed that only the CEV samples had normal amplification, and the negative control (DEPC-treated water) and KHV, GFHNV, EHNV, BIV, CCV, WSSV, and EHP samples had no amplification (such as image 3 shown). The above results indicate that the RAA-LFD rapid detection kit of the present invention can specifically amplify the target sequence in CEV without cross-reaction with other viral nucleic acids. It shows that the method and kit of the present invention have good specificity.

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Abstract

The invention discloses a RAA amplification primer and a probe for rapid detection of Cyprinid herpesvirus II, a detection kit and an application method thereof. The RAA isothermal amplification system of the invention has fast reaction and wide temperature range, can realize effective amplification of target genes at 34-40 DEG C, has a lowest detection limit of 8.14*100 copies / microliter, has a higher sensitivity than common PCR of CyHV-2, and will not cross-react with other aquatic animal epidemics. The kit of the invention can quickly, efficiently and sensitively detect Cyprinid herpesvirus II, is simple to operate, has high specificity, is easy to observe reaction results, and is safe and pollution-free. The invention not only provides an effective technical means for on-site rapid detection and screening of Cyprinid herpesvirus type II infection nucleic acid, but also has very important significance for controlling the infection transmission of the virus in ornamental fish such as crucian carp and goldfish and the inspection and quarantine in epidemic areas and entry and 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 herpesvirus type II, a detection kit and a use method. Background technique [0002] Cyprinid herpesvirus Ⅱ (Cyprinid herpesvirus 2, CyHV-2) is a highly pathogenic pathogen that infects goldfish and crucian carp (Carassius auratus) and causes necrosis of hematopoietic organs, seriously endangering the fish farming industry of goldfish and crucian carp. The virus infects the gills, kidneys, and spleen tissues of fish. Infected fish are sluggish, have enlarged abdomen, and bleed on the body surface and at the base of fin rays. In 1995, the virus was first isolated from goldfish raised in Japan, and then spread rapidly to the United States, Australia, the United Kingdom and other countries and Taiwan. After the virus was introduced into my country, a large-scale outbreak occurred in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/705C12Q1/6844
Inventor 吕晓楠王姝徐立蒲张文曹欢王静波王小亮
Owner 北京市水产技术推广站
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