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Primer and TaqMan probe for qualitative and quantitative detection of blue tongue virus

A technology for quantitative detection of bluetongue virus, applied in the direction of microbial determination/inspection, biochemical equipment and methods, 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: 2006-12-20
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 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 and TaqMan probe for qualitative and quantitative detection of blue tongue virus
  • Primer and TaqMan probe for qualitative and quantitative detection of blue tongue virus
  • Primer and TaqMan probe for qualitative and quantitative detection of blue tongue virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, be used for bluetongue virus (BTV) carrying out the design of the primer of qualitative, quantitative detection and TaqMan probe

[0034] 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, DNAStar software was used to analyze The 6 serotypes / strain BTV NS1 gene sequences were compared, and then primers and TaqMan probes were designed according to the comparison results. The sequences are as follows:

[0035] Upstream primer P (NS1-1): 5'-GTTTCTAGTTGGCAACCACC-3' (SEQ ID NO: 1 in the sequence listing), Tm is 57°C;

[0036] Downstream pr...

Embodiment 2

[0043] Embodiment 2, carry out the conventional PCR detection of BTV to bluetongue virus with primers of the present invention

[0044] 1. Processing of BTV samples

[0045] 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.

[0046] The cell-cultured BTV samples were processed by the following method: first inoculate BTV to BHK-21 cells, and inoculate at 37°C CO 2 Cultivate in an incubator until the pathological changes of the cells reach more than 80%, then transfer the diseased cells into a sterilized centrifuge tube under aseptic conditions, centrifuge at 8000rpm for 30min, and transfer the supernatant to another centrifuge tube under aseptic conditions and temporarily place it at room temperature Next, the cell pellet was repeatedly frozen and thawed three times, then the supernatant was added, mixed by pipetting, centrifuged at 8000rpm for 30min, and the supernatan...

Embodiment 3

[0054] 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

[0055] 1. Cultivation and treatment of BTV

[0056] 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.

[0057] 2. Extraction of BTV RNA

[0058] Take 300 μl of the BTV sample obtained in step 1, and use the same method as in Example 2 to extract RNA.

[0059] 3. Reverse transcription

[0060] 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.

[0061] 4. Preparation of real-time FQ PCR standards

[0062] (1) PCR amplification product recovery of BTV-5

[0063] 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 qualitative, quantitative detected primer and TaqMan probe of bluetongue virus, which is characterized by the following: the upstream primer of primer possesses SEQ ID No.:1 in the sequence list and downstream primer possesses SEQ ID No.:2 in the sequence list; the TaqMan probe possesses SEQ ID No.:3 or SEQ ID NO:4 DNA sequence in the sequence list; the 5'-end of probe marks report fluorescent group and 3'-end marks quenched fluorescent group; fitting for clinical and scientific study to proceed qualitative and quantitative analysis for bluetongue RNA; possessing important meaning to evaluate and observe bluetongue happening, recurrence and treating effect.

Description

technical field [0001] 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 [0002] 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 world. Since the first occurrence of the disease in Shizong County, Yunnan Province in 1979 in my country, it has occurred repeatedly in other areas. [0003] Bluetongue ...

Claims

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Application Information

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