Dual-fluorescence quantitative RT-PCR primers and probes for identifying and detecting rift valley fever virus

A technology of Rift Valley fever virus and dual fluorescence, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of increasing dosage, save manpower and material resources, avoid false positive results, and operate simple effect

Inactive Publication Date: 2019-10-01
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although inactivated vaccines are safe, they must be dosed repeatedly; although attenuated live vaccines have high immunogenicity, there is a biosafety risk of virulence regression

Method used

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  • Dual-fluorescence quantitative RT-PCR primers and probes for identifying and detecting rift valley fever virus
  • Dual-fluorescence quantitative RT-PCR primers and probes for identifying and detecting rift valley fever virus
  • Dual-fluorescence quantitative RT-PCR primers and probes for identifying and detecting rift valley fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Primer probe design and method establishment

[0042] 1. Design of primers and probes: Through comparative analysis of Rift Valley fever virus genome sequences, select S segment NSs gene, N gene and L gene with no secondary structure and highly conserved segments, and use Primer Express 3.0 software to design respectively More than one hundred combinations of primers and probes were selected, and the effect of the primer combinations was evaluated by using the MPprimer software of the multiplex PCR primer design system, and then the reaction conditions were verified and optimized through experiments, in order to screen the best combination of target genes, primers and probes, and at the same time Two genes are tested. In the end, only one primer-probe combination was selected from more than one hundred primer and probe combinations to achieve good amplification of two genes. The optimal primer and probe sequence combination is as follows:

[0043] The sequ...

Embodiment 2

[0125] Embodiment 2 specific detection

[0126] Rift Valley fever virus positive plasmids and other pathogenic nucleic acids including Peste des Petits Ruminants (PPRVNigeria 75 / 1 vaccine strain, purchased from Xinjiang Tiankang Animal Husbandry Biotechnology Co., Ltd.), goat parainfluenza virus type 3 (JS2013 strain, Li Wenliang et al. Isolation and molecular identification of goat-derived parainfluenza virus type 3. Journal of Animal Husbandry and Veterinary Medicine, 2015, 46(2): 344-348), bluetongue virus (75-1 strain, Li Wenliang et al. , Isolation and Identification of Serum Type 15 and Type 21 Bluetongue Viruses. Southwest Agricultural Journal, 2018, 31(3): 630-634), Goat Herpes Virus Type I (JSHA1405 strain, Hao Fei et al., Goat Herpes Virus Type I Isolation and identification. Animal Husbandry and Veterinary Journal, 2018,49(8):1797-1802), Mycoplasma ovis pneumoniae (Y98 strain, purchased from China Veterinary Drug Control Institute) were tested to verify whether the ...

Embodiment 3

[0128] The mensuration of embodiment 3 sensitivity

[0129] Separate the two positive plasmid standards from 1×10 8 copies / μL serially diluted to 1×10 1 copy / μL, and then detect according to the amplification system and conditions described in the examples, and determine the lowest detection limit of the method of the present invention.

[0130] The result is as Figure 5 As shown, Figure A is the sensitivity amplification curve of the NSs gene standard product, and Figure B is the sensitivity amplification curve of the L gene standard product. It can be seen that under the optimal reaction conditions, the method of the present invention can produce typical amplification curves for the amplification of template amounts above 10 copies / μL.

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Abstract

The invention provides dual-fluorescence quantitative RT-PCR primers and probes for identifying and detecting rift valley fever virus and belongs to the field of biotechnology. The pair of specific detection primers L-F and L-R and the fluorescent probe L-P for an L gene of rift valley fever virus and the pair of specific detection primers S-F and S-R and the fluorescent probe S-P for an S fragment of rift valley fever virus are designed, and the concentration and reaction conditions of the specific primers and fluorescent probes in a method are optimized. The provided dual-fluorescence quantitative RT-PCR method can be used for detecting the infection of rift valley fever virus and meanwhile identifying rift valley fever virus wild strains and NSs deletion strains including Clone 13 and other deletion strains, so that infected animals are distinguished from immunized animals. The primers and the probes have the advantages of simple operation, high sensitivity and specificity, good repeatability and the like and can not only reduce the cost but also gain valuable time for epidemic detection and control.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a double fluorescent quantitative RT-PCR primer and probe for identifying and detecting Rift Valley fever virus. Background technique [0002] Rift valley fever (RVF) is an acute hemorrhagic zoonotic disease caused by Rift valley fever virus (RVFV), which mainly occurs in sheep, goats, cattle and camels, and can also Infects rodents and humans. The Rift Valley fever virus is mainly transmitted by mosquitoes, and can also be infected through contact with the blood, body fluids, excretions, etc. of infected or diseased animals. The severe symptoms of Rift Valley fever in animals are fever, anorexia, runny nose, acute diarrhea, jaundice, the mortality rate of lambs and calves is as high as 90%, and the abortion rate of pregnant ewes and cows is as high as 80-100%. Human infection is usually asymptomatic or mildly symptomatic, and less than 5% of patients develop syndrom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2531/113C12Q2545/114
Inventor 杨蕾蕾李文良毛立郝飞李基棕张纹纹江杰元孙敏刘茂军
Owner JIANGSU ACAD OF AGRI SCI
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