Fluorescent quantitative reverse-transcription-polymerase chain reaction (RT-PCR) primer for detecting schmallenberg virus and probe

A fluorescent quantitative and probe technology, applied in the field of fluorescent quantitative RT-PCR detection, can solve the problem of not completely ruling out the occurrence of Schmallenberg virus in humans, avoiding false negative results, saving manpower and material resources, and achieving good specificity. Effect

Inactive Publication Date: 2013-09-18
CHINESE ACAD OF INSPECTION & QUARANTINE
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Problems solved by technology

Given that most viruses of the Bunyaviridae have the characteristics of zoonosis and the same transmission medium (mainly Culicoides,

Method used

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  • Fluorescent quantitative reverse-transcription-polymerase chain reaction (RT-PCR) primer for detecting schmallenberg virus and probe
  • Fluorescent quantitative reverse-transcription-polymerase chain reaction (RT-PCR) primer for detecting schmallenberg virus and probe
  • Fluorescent quantitative reverse-transcription-polymerase chain reaction (RT-PCR) primer for detecting schmallenberg virus and probe

Examples

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Example Embodiment

[0030] Example 1 Fluorescence quantitative RT-PCR detection of Schmallenberg virus

[0031] 1 Design of primers and probes: by comparing and analyzing the genome sequences of Bunyaviridae and Schmallenberg virus published in GenBank, selecting highly conserved segments without secondary structure and designing multiple pairs For primers and probes, the length of the primer is generally about 25 bases, and there is no complementary sequence between and within the primer. The optimal primers and probes (designed according to the S gene) sequence combination are as follows (SEQ ID No.1-3):

[0032] Upstream primer SBV-S-130F: 5’-GAAGCTAGTGCTCAGATTGTCATGC-3’

[0033] Downstream primer SBV-S-130R: 5’-GTGGATAGAAGTCAAAAGCATCAAGG-3’

[0034] TaqMan probe SBV-S-130FAM:

[0035] 5’-FAM-AAGGGATGCACCTGGGCCGATGGTTA-BHQ1-3’

[0036] 2 Preparation of Schmallenberg virus RNA: select the primer pair SBV-S-130F / R and probe SBV-S-130FAM, and extract the Schmallenberg virus RNA from the sample tissue to b...

Example Embodiment

[0082] Example 2 Specific analysis of Schmallenberg virus fluorescence quantitative RT-PCR detection

[0083] The 12 copies of the Bunyavirus family Simbu serogroup (Simbu serogroup) preserved by the German FLI Research Institute are closest to SBV. Sango Texas, Shuni Texas, Aino Texas, Simbu Texas, Peaton Texas, Satuperi Texas, Akabane Australien A347, Akabane Boeringer Vakzine A, Oropouche Australien O, AINO Z445Australien, Tinaroo A371Australien and Thimiri V981Australien virus RNA samples and 132 SBV-negative cattle and sheep RNA samples, as well as 46 cattle and 46 sheep negative serum RNA samples collected from China, verified The specificity of primers and probes for Schmallenberg virus detection. The results showed that all the above-mentioned SBV-negative RNA samples were amplified by fluorescence quantitative RT-PCR, and there was no specific amplification curve, only SBV RNA standard products (10 -3 ) The typical amplification appears, indicating that the designed prim...

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Abstract

The invention provides a fluorescent quantitative reverse-transcription-polymerase chain reaction (RT-PCR) primer for detecting schmallenberg virus and a probe. The nucleotide sequences of the primer and the probe are as shown in SEQ ID No.1-3. The sensitivities of the primer and the probe provided by the invention can reach 10<-7> of a schmallenberg virus (SBV) ribose nucleic acid (RNA) standard substance; and moreover, the primer and the probe do not have amplification signals on detection samples without schmallenberg virus, so that the good specificities of the primer and the probe are shown.

Description

technical field [0001] The invention relates to a fluorescent quantitative RT-PCR detection technology, in particular to a fluorescent quantitative RT-PCR primer and probe for detecting Schmallenberg virus. Background technique [0002] In the summer and autumn of 2011, an unknown disease began to appear in some cattle farms in Germany and the Netherlands. The sick cows showed clinical symptoms such as fever (>40°C), physical decline, loss of appetite, anorexia, diarrhea, and decreased milk production. miscarriage symptoms. On November 18, 2011, the Friedrich Loeffler Institute (FLI) in Germany used Metagenomics technology (Metagenomic technique) and virus isolation technology to finally confirm that the pathogen that caused the outbreak was a new type of Bunia virus. Because the virus was first isolated and identified from the blood of a sick cow in Schmallenberg, a town in western Germany, it was tentatively named "Schmallenberg virus (SBV)" according to the place of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 张永宁吴绍强林祥梅吕继洲王彩霞邓俊花袁向芬
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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