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Primers and probe for detecting fragment S of rift valley fever virus

A Rift Valley fever virus and probe technology, applied in the field of genetic engineering, can solve problems such as differences in pathogenicity of virus strains, and achieve the effects of high sensitivity, high sensitivity, and rapid response

Inactive Publication Date: 2013-05-15
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, no specific antigenic differences have been found between RVFV isolates and laboratory passage strains, but it has been proved that there are certain differences in the pathogenicity of each strain

Method used

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  • Primers and probe for detecting fragment S of rift valley fever virus
  • Primers and probe for detecting fragment S of rift valley fever virus
  • Primers and probe for detecting fragment S of rift valley fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The preparation of embodiment 1 primer and probe

[0020] 1.1 Design and synthesis of primers and probes

[0021] The conserved sequence of the S segment of Rift Valley fever virus was retrieved from the NCBI Gene Bank (GenBank) of the National Center for Bioinformatics in the United States, and the accession number was (DQ380149). The gene sequence was analyzed by MegAlign software, and according to the design principles of primers and probes, a pair of primers were screened in the conserved region of the sequence with BeaconDesigner 5.0, and a fluorescent TaqMan probe was set in the amplified region of the primer pair.

[0022] The primer sequences are:

[0023] RVFVU1: 5'-GGATTACTTTCCTGTGATATCTGTTG-3'

[0024] RVFVL1: 5'-GTATCCTGGGAGGRCCATCWC-3' (R is A or G, W is T or A), the length of the amplified product is 92bp;

[0025] The sequence of the probe (RVFVP1) is: 5'-ACTCCACTGACACAACACGACGACCACT-3', the 5' end of the probe is labeled with the reporter fluorescent ...

Embodiment 2

[0028] The establishment of embodiment 2 fluorescent PCR detection method

[0029] 1.1 Preparation of Rift Valley fever S segment recombinant plasmid

[0030] The conserved sequence of the Rift Valley fever virus S segment was retrieved from the NCBI Gene Bank (GenBank) of the National Center for Bioinformatics in the United States. After chemical synthesis, a restriction site (KpnI and HindIII) was introduced at both ends, and then ligated into the cloning vector pGEM In -T, transform Escherichia coli competent BL21, and select white spot shake bacteria after blue-white screening. The positive bacterial solution is sequenced and the plasmid and recombinant plasmid (pGEM-T-rvfv-s) are extracted. The measured concentration is 1.0×10 8 copy / μL, prepared as a standard plasmid containing the conserved sequence of the S segment of Rift Valley fever virus. The plasmid was sequentially diluted 10 times to 1.0×10 with Dilution buffer (TaKaRa). 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 ,...

Embodiment 3

[0038] Example 3 Sensitivity test of Rift Valley fever virus S segment real-time fluorescent PCR method

[0039] Using the standard plasmid prepared in Example 2, the experiment was carried out using the real-time fluorescent PCR reaction system for S segment of Rift Valley fever virus established above. After the reaction is finished, the instrument automatically displays the amplification curve ( figure 1 ). figure 1 The amplification curves and CT values ​​of different copies of plasmids are shown, and the detection CT value of 10 copies of plasmids is 37.62, so it can be judged that the sensitivity of this method can reach 10 copies of plasmid DNA.

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Abstract

The invention provides primers and a probe for detecting the fragment S of a rift valley fever virus, wherein, a forward primer is 5'-GGATTACTTTCCTGTGATATCTGTTG-3'; a reverse primer is 5'-GTATCCTGGGAGGRCCATCWC-3', R refers to A or G and w refers to T or A); and a probe is 5'-F1-ACTCCACTGACACAACACGACGACCACT-Q1-3', F1 is a fluorescent reporter and Q1 is a fluorescent quencher. The invention also provides a real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection method for the fragment S of the rift valley fever virus. In the method, pseudovirus grains of the rift valley fever virus are taken as a template, and the primers and the probe are used to carry out real-time fluorescent RT-PCR, thus judging whether the detected virus is the rift valley fever virus according to the detected result. The probe provided by the invention has high sensitivity and good specificity, and has no cross reaction with all of Africa swine fever virus, irides virus and goatpox virus.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a primer and a probe for detecting the S segment of Rift Valley fever virus. Background technique [0002] Rift valley fever (Rift valley fever, RVF) is a serious zoonotic disease and an endemic disease that has been prevalent in the Grand Canyon of East Africa since ancient times. It is mainly transmitted among sheep, goats and cattle, and can often cause abortion in pregnant animals. Storm (abortion storm), young sheep mortality rate as high as 90%. The epidemic cycle of the disease is about 5-10 years. The disease can infect humans, and the patients show flu-like symptoms such as fever, headache and arthralgia. A few have hemorrhagic fever and encephalitis, and occasionally can cause retinitis and even blindness. The case fatality rate is about 1%. Up to about 50%. RVF is mainly transmitted by mosquitoes, has obvious seasonality, and can also be infected by contact. [0...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 王彩霞林祥梅吴绍强张永宁邓俊花
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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