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Primer for quantitative and qualitative determination of blue tongue virus

A technology for quantitative detection of bluetongue virus, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of real-time fluorescent quantitative PCR detection technology without bluetongue virus gene, without primers and Specific fluorescent probes and other issues

Active Publication Date: 2008-10-08
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, there is currently no real-time fluorescent quantitative PCR detection technology specifically for bluetongue virus genes, because there are no suitable primers and specific fluorescent probes

Method used

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  • Primer for quantitative and qualitative determination of blue tongue virus
  • Primer for quantitative and qualitative determination of blue tongue virus
  • Primer for quantitative and qualitative determination of blue tongue virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, be used for bluetongue virus (BTV) carrying out the design of the primer of qualitative, quantitative detection and TaqMan probe

[0035] According to the NS1 gene sequences of 8 serotypes of BTV in GenBank, DNAStar software was used to compare the sequences of the NS1 genes of the 8 serotypes of BTV, and the most conserved region sequence at the 5' end of the NS1 gene was selected as the detection sequence. Since there are 6 serotype BTV NS1 gene sequences (GenBankAccession No.AY462225, M97762, NC 006025, X15891, X56735, Y00422) in the 8 serotype BTV NS1 genes including a longer 5' non-coding region sequence, using DNAStar software The 6 serotypes / strains of BTV NS1 gene sequences were compared, and then primers and TaqMan probes were designed according to the comparison results. The sequences are as follows:

[0036] Upstream primer P (NS1-1): 5'-GTTTCTAGTTGGCAACCACC-3' (SEQ ID NO: 1 in the sequence listing), Tm is 57°C;

[0037] Downstream primer P (NS...

Embodiment 2

[0044] Embodiment 2, carry out the conventional PCR detection of BTV to bluetongue virus with primers of the present invention

[0045] 1. Processing of BTV samples

[0046] Eight BTV serotypes (BTV3, BTV5, BTV8, BTV10, BTV11, BTV21, BTV22, BTV(A)) in cell cultured BTV, plasma and tissue were selected as BTV samples to be tested.

[0047] The following method is used to process the BTV samples cultured by cells: first inoculate BTV to BHK-21 cells, cultivate them in a CO2 incubator at 37°C until the pathological changes of the cells reach more than 80%, and then transfer the pathological cells into In a sterilized centrifuge tube, centrifuge at 8000rpm for 30min. Under sterile conditions, transfer the supernatant to another centrifuge tube and temporarily place it at room temperature. After repeated freezing and thawing of the cell pellet three times, add the supernatant, blow and mix, and centrifuge at 8000rpm for 30min. The supernatant was used for the extraction of BTV RNA...

Embodiment 3

[0055] Embodiment 3, carry out the real-time fluorescent quantitative PCR (real-timeFQ PCR) detection of BTV with primer of the present invention and TaqMan probe

[0056] 1. Cultivation and treatment of BTV

[0057] Cell-cultured BTV was selected as the BTV sample to be tested, and the same method as in Example 2 was used to treat the cell-cultured BTV sample.

[0058] 2. Extraction of BTV RNA

[0059] Take 300 μl of the BTV sample obtained in step 1, and use the same method as in Example 2 to extract RNA.

[0060] 3. Reverse transcription

[0061] Using the RNA of the BTV sample extracted in step 2 as a template, its cDNA was synthesized by reverse transcription, and the reaction system and reaction conditions were the same as those in Example 2.

[0062] 4. Preparation of real-time FQ PCR standards

[0063] (1) PCR amplification product recovery of BTV-5

[0064] Take 50 μl of the PCR amplification product of the detection sample BTV5 obtained in Example 2, carry out 2...

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Abstract

The invention discloses a primer used for blue tongue virus qualitative and quantitative measurement. The upstream primer of the primer is provided with SEQ ID NO. 1 in the sequence table and the downstream primer of the primer is provided with SEQ ID NO. 2 in the sequence table. The primer can be used for qualitative and quantitative analyzing of the blue tongue virus ribonucleic acid of the animals which is infected with the blue tongue virus in clinic and scientific researches, and the primer is provided with significant meaning to the judgment of occurring and recurring of the blue tongue virus, the curing effect evaluation and the dynamic observation of the patients condition; the invention can play an important role in the animal medicine detecting field.

Description

[0001] The present invention is a divisional application of the patent application with the application date of May 16, 2006 and the application number 200610081309.3. technical field [0002] The invention relates to primers and probes for qualitative and quantitative detection of viruses with molecular biological methods, in particular to real-time fluorescence quantitative PCR (real-time fluorescent quantification PCR, real-time FQPCR) technology for bluetongue virus qualitative and quantitative detection. Primers and TaqMan probes for quantitative detection. Background technique [0003] Bluetongue is one of the 15 Class A animal diseases recognized by the World Organization for Animal Health (OIE), and has received special attention from relevant countries around the world. Bluetongue disease first occurred in South Africa (in the late 19th century). After entering the 20th century, the disease occurred or the disease virus was detected one after another all over the wo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 章金刚尹惠琼
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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