Unlock instant, AI-driven research and patent intelligence for your innovation.

General type RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection primers for grass carp reoviruses of different genotypes and application of general type RT-PCR detection primers

An RT-PCR and reovirus technology, which is applied in the field of universal RT-PCR detection primers for different genotypes of grass carp reovirus, can solve the problems of increased detection cost and prolonged detection time, and achieves reduction of detection cost and sensitivity. High, specific effect

Active Publication Date: 2018-07-31
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
View PDF3 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • General type RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection primers for grass carp reoviruses of different genotypes and application of general type RT-PCR detection primers
  • General type RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection primers for grass carp reoviruses of different genotypes and application of general type RT-PCR detection primers
  • General type RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection primers for grass carp reoviruses of different genotypes and application of general type RT-PCR detection primers

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 GCRVI, II and III are all about 590bp, of which GCRVI is 590bp, GCRVII is 590bp or 593bp, and GCRVIII is 587bp.

[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 kind of different genotype grass carp reovirus universal type RT-PCR detection method, comprises the following steps:

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

[0034] For the extraction of nucleic acid from suspected samples of grass carp hemorrhagic disease, the liver, spleen, kidney and other visceral tissues of the diseased fish were collected in a sterile petr...

Embodiment 3

[0041] Detection of primer specificity:

[0042] Using the method described in Example 2 to detect channel catfish reovirus (CCRV, highly homologous to type I), giant salamander iridescent virus (GSIV), koi herpes virus (KHV), carp herpes virus type II (CyHV-2 ), carp spring viremia virus (SVCV) and infectious spleen-kidney necrosis virus (ISKNV), and the above 13 grass carp reoviruses. , take 5 μL of the PCR amplification product, and use 1.0% agarose gel electrophoresis to detect.

[0043] The results of electrophoresis detection show that the cDNA of three genotypes of grass carp reovirus and channel catfish reovirus (CCRV) can be amplified to obtain the target band of about 590bp in expected size, and the sequencing results show that the actual length Consistent with that described in Example 2; And all can not amplify any band with GSIV, KHV, CyHV-2, SVCV and ISKNV etc. other fish common virus nucleic acid or reverse transcription product as template (partial result is a...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 molecular biology detection of fish viruses, and in particular relates to a universal RT-PCR detection primer for different genotypes of grass carp reovirus and its application. Background technique [0002] Grass carp reovirus (GCRV) is the first fish virus isolated in my country. The diameter of the virus particle is about 60-70nm, icosahedral symmetry, double-layer capsid, no envelope, and the genome consists of 11 segmented double-stranded RNAs. Grass carp haemorrhagic disease caused by grass carp reovirus is extremely harmful to grass carp, the main freshwater species in my country. The economic loss caused by the disease reaches more than one billion yuan every year, seriously affecting the healthy development of grass carp aquaculture in my country. At present, there are more than 30 GCRV isolates reported in my country, which can be divided into three genotypes according to the differences in gene sequence...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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