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A universal rt-PCR detection primer for grass carp reovirus with different genotypes and its application

A technology of RT-PCR and reovirus, which is applied in the field of general-purpose RT-PCR detection primers for different genotypes of grass carp reovirus, can solve the problems of increased detection cost and prolonged detection time, and achieve lower detection cost and higher sensitivity High and specific effect

Active Publication Date: 2020-09-29
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To cover the current three genotypes of GCRV, it is necessary to design multiple pairs of primers or multiple reactions to complete, prolonging the detection time and increasing the detection cost
Especially in recent years, there have been reports of GCRV isolates from different places one after another, and some new isolates have large differences in gene sequence from existing isolates. Therefore, it is particularly necessary to comprehensively analyze the sequences of existing GCRV isolates and establish a simple, fast and universal RT-PCR detection method applicable to different genotypes of GCRV isolates.

Method used

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  • A universal rt-PCR detection primer for grass carp reovirus with different genotypes and its application
  • A universal rt-PCR detection primer for grass carp reovirus with different genotypes and its application
  • A universal rt-PCR detection primer for grass carp reovirus with different genotypes and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Obtaining degenerate primers:

[0026] For 13 GCRV isolates, 2 strains of type Ⅰ, 10 strains of type Ⅱ, and 1 strain of type Ⅲ, degenerate primers were designed:

[0027] GCRV-dpF: 5’–TAYGTVACMSCCMGRGGWGG–3’;

[0028] GCRV-dpR: 5’–AADTGYTGYACCATGDYCTGC–3’;

[0029] The size of the fragments amplified by the above primers in GCRV I, II and III are all about 590 bp, of which GCR I is 590 bp, GCRV II is 590 bp or 593 bp, and GCRV III is 587 bp.

[0030] The specific strains are as follows: GCRV-873 (type I, AF284502.1), GCRV-GZ1208 (type I, KU240075.1), GCRV-HZ08 (type II, GQ896335.1), GCRV-GD108 (type II, HQ231199. 1), GCRV106 (type Ⅱ, KC201167.1), GCRV918 (type Ⅱ, KC201178.1), GCRV-JX02 (type Ⅱ, KM880066.1), GCRV-HuNan794 (type Ⅱ, KC238677.1), GCRV-Huan1307 (Type Ⅱ, KU254567.1), GCRV-AH528 (Type Ⅱ, KR180369.1), GCRV-HeNan988 (Type Ⅱ, KC847321.1), GCREV109 (Type Ⅱ, KF712476.1), GCRV104 (Type Ⅲ, JN967630. 1).

Embodiment 2

[0032] A universal RT-PCR detection method for different genotypes of grass carp reovirus, including the following steps:

[0033] 1) Viral nucleic acid extraction: Collect virus-infected cells, freeze-thaw repeatedly at -80°C and room temperature for 3 times, centrifuge at 4000r / min for 30min (Sigma, 3K-15), transfer the supernatant to a 35mL ultracentrifuge tube, Centrifuge at 20 000 r / min for 2 h (Optima L-80XP, Beckman-Coulter), and suspend the virus pellet for use. RNA virus and DNA virus nucleic acid were extracted with Trizol and DNAzol reagents from Invitrogen, and the specific operations were carried out according to the reagent instructions. The extracted viral RNA and DNA were stored in the refrigerator at -80°C and -20°C for later use.

[0034] For the nucleic acid extraction of the collected samples of suspected grass carp hemorrhagic disease, collect the liver, spleen, kidney and other internal organs of the diseased fish in a sterile petri dish, cut them with steril...

Embodiment 3

[0041] Primer specific detection:

[0042] The method described in Example 2 was used to detect channel catfish reovirus (CCRV, highly homologous to type I), giant salamander iridescent virus (GSIV), koi herpes virus (KHV), and carp herpes virus type II (CyHV-2) ), Carp Spring Viremia Virus (SVCV) and Infectious Spleen and Kidney Necrosis Virus (ISKNV), as well as the aforementioned 13 grass carp reoviruses. , Take 5μL of PCR amplification product, use 1.0% agarose gel electrophoresis to detect.

[0043] The results of electrophoresis showed that the cDNA of the three genotypes of grass carp reovirus and channel catfish reovirus (CCRV) can be amplified to obtain the expected size of about 590bp bands. The sequencing results show that its actual length It is consistent with the description in Example 2. However, using GSIV, KHV, CyHV-2, SVCV and ISKNV and other common fish viral nucleic acids or reverse transcription products as templates cannot amplify any bands (part of the resul...

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Abstract

The invention discloses general type RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection primers for grass carp reoviruses of different genotypes and application of the general type RT-PCR detection primers. A method disclosed by the invention only uses a pair of primers and performs PCR reaction once to simultaneously detect grass carp reoviruses of three genotypes (type I, type IIand type III), thereby greatly improving the detection efficiency and reducing the detection cost; and furthermore, the method also has the advantages of high sensitivity, strong specificity and thelike and can play important roles in rapid diagnosis and epidemiological investigation of clinical samples of grass carp with bleeding diseases.

Description

Technical field [0001] The invention belongs to the field of fish virus molecular biology detection, and specifically relates to a universal RT-PCR detection primer for grass carp reovirus of different genotypes and its application. Background technique [0002] Grass carp reovirus (grass carp reovirus, GCRV) is the first fish virus isolated in my country. The virus particle has a diameter of about 60-70 nm, icosahedral symmetry, a double-layer capsid, no envelope, and the genome is composed of 11 segments of double-stranded RNA. The grass carp hemorrhagic disease caused by the grass carp reovirus is extremely harmful to the main freshwater species of grass carp in my country. The economic loss caused by the disease reaches hundreds of millions of yuan each year, which seriously affects the healthy development of my country's grass carp breeding industry. At present, there are more than 30 GCRV isolates reported in my country, which can be divided into 3 genotypes according to g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2531/113
Inventor 范玉顶曾令兵周勇江南刘文枝
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI