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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Given that most viruses of the Bunyaviridae have the characteristics of zoonosis and the same transmission medium (mainly Culicoides, mosquitoes and sandflies), the possibility that Schmallenberg virus can cause human disease cannot be completely ruled out at present

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

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Effect test

Embodiment 1

[0030] Fluorescent quantitative RT-PCR detection of embodiment 1 Schmallenberg virus

[0031] 1 Design of primers and probes: By comparing and analyzing the genome sequences of related viruses of the Bunyaviridae family and Schmallenberg virus published in GenBank, a segment with no secondary structure and a high degree of conservation was selected, and multiple pairs were designed. For primers and probes, the length of the primers is generally about 25 bases, and there is no complementary sequence between and within the primers. The sequence combination of optimal primers and probes (designed according to the S gene) is 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 primer pair SBV-S-130F / R and probe SBV-S-130...

Embodiment 2

[0082] Example 2 Specificity Analysis of Schmallenberg Virus Fluorescent Quantitative RT-PCR Detection

[0083] Sango Texas, Shuni Texas, Aino Texas, Simbu Texas, Peaton Texas, Sathuperi 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 Specificity of primers and probes for detection of Schmallenberg virus. The results showed that all the above-mentioned SBV negative RNA samples were amplified by fluorescent quantitative RT-PCR, and no specific amplification curve appeared, only the SBV RNA standard (10 -3 ) showed typical amplification, indicating that the designed primers and probes had good specificity for Schmallenberg virus.

[0084] image 3 It is a fluorescent quantitative RT-qPCR amplification curve for ...

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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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