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Nested PCR kit and method for detecting double-RNA viruses of micropterus salmoides

An RNA virus and largemouth bass technology, which is applied in the field of nested PCR kits for detecting largemouth bass double RNA virus, can solve the problems of publication and no virus detection method, and achieves the goal of increasing revenue, eliminating the risk of virus transmission, and reducing losses. Effect

Active Publication Date: 2021-02-05
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

And the object of the present invention is an unknown aquatic animal double RNA virus, so far, there is no publication about the detection method of this kind of virus

Method used

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  • Nested PCR kit and method for detecting double-RNA viruses of micropterus salmoides
  • Nested PCR kit and method for detecting double-RNA viruses of micropterus salmoides
  • Nested PCR kit and method for detecting double-RNA viruses of micropterus salmoides

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Effect test

Embodiment 1

[0034] Embodiment 1 Materials and methods

[0035] 1.1 Cells and virus strains

[0036] The Chinese perch brain cell line (CPB) was established and preserved in our laboratory. Largemouth bass birnavirus (LBBV), grouper neuronecrosis virus (Nervous Necrosis Virus, NNV), carp herpesvirus II (Cyprinid herpesvirus II, CyHV-II), mandarinfish ranavirus (MRV) ), grouper iridovirus (SGIV), giant salamander iridovirus (Andrias davidianus iridovirus, ADIV), mandarin fish infectious spleen and kidney necrosis virus (ISKNV), mandarin fish bullet virus (Sinipercachuatsis rhabdovirus, SCRV) and Tilapia Lake Virus (TiLV) were isolated and preserved by our laboratory.

[0037] 1.2 Method

[0038] 1.2.1 Primer design and synthesis

[0039] According to the VP1 sequence of LBBV (the nucleotide sequence is shown in SEQ ID NO.1), using Primer 5.0 software, VP1 gene amplification primers (for plasmid construction) and nested PCR primers (see Table 1) were designed. The primer sequences were ...

Embodiment 2

[0067] Embodiment 2 result and analysis

[0068] 2.1 Primer condition optimization

[0069] The result is as figure 1 As shown, when the addition amount of primers F1 / R1 and F2 / R2 was 4 pmol respectively, the amplified product had the brightest band and no primer-dimer. When the primer amount was greater than 4 pmol, the primer-dimer Increased with increasing, and the brightness of the target band weakened, so the optimal primer addition amount was 4pmol.

[0070] 2.2 Annealing temperature optimization

[0071] Such as figure 2 (A) shows that the optimal annealing temperature for F1 / R1 is 64.1°C. When the temperature is higher or lower than 64.1°C, the amplification efficiency will gradually decrease, and the bands will gradually become darker. When the temperature is lower than 50.4°C, non-specific amplification will occur; figure 2 (B) shows that the optimal annealing temperature for F2 / R2 is 61.5°C. When the temperature is higher or lower than 61.5°C, the amplificatio...

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Abstract

The invention relates to the technical field of double-RNA virus detection, in particular to a nested PCR kit and method for detecting double-RNA viruses of micropterus salmoides. The kit comprises two pairs of nested PCR primers, wherein the sequences of the two pairs of nested PCR primers are that F1 of 5'-CAGAAGGACCGATTCAACTCACT-3', R1 of 5'- CTCTGGTGAGGAGGTAGTAGGCAA-3', F2 of 5'-CCTGTCGTGCGGGCTCCTATT-3', and R2 of 5'- CTCTTTGTGGCGTTGGCTTCG-3'. According to the nested PCR kit, aiming at LBBV, the VP1 conserved gene sequence of LBBV is taken as a target to establish a nested PCR detection method, and a quick and sensitive detection means is provided for early warning and effective prevention and control of the virus disease.

Description

technical field [0001] The invention relates to the technical field of double RNA virus detection, in particular to a nested PCR kit and method for detecting largemouth bass double RNA virus. Background technique [0002] The largemouth bass (Micropterus salmoides) belongs to the family Centrarchidae and the genus Micropterus. The meat is delicious and tender, without intermuscular thorns, and rich in nutrition. It is a high-quality freshwater fish. The inventor isolated a new strain of double RNA virus from the tissue of the diseased largemouth bass. The main clinical symptoms were yellow mucus in the intestine, hemorrhage in the liver, and ascites; the virus had no envelope and had an icosahedral structure; the genome consisted of a double Segmented double-stranded RNA, segment A encodes four proteins of VP2, VP4, and VP3, respectively, and segment B encodes VP1 protein; gene homology analysis shows that the virus is similar to Blosnavirus genus of the Birnaviridae family...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6848C12Q2549/119C12Q2565/125Y02A40/81
Inventor 付小哲李宁求罗明菊林强刘礼辉牛银杰罗霞梁红茹
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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