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Primer probe group and kit for combined detection of Batai virus and Taina virus based on dual fluorescence PCR method

A technology of Batay virus and primer probe, which is applied in the field of primer-probe group for joint detection of Batay virus and Tyna virus based on dual fluorescent PCR method, can solve the problems of lack of effective detection methods for Batay virus and Tyna virus

Pending Publication Date: 2018-12-07
广东省公共卫生研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The invention provides a primer probe set, a kit and a method of use thereof based on a dual fluorescent PCR method for joint detection of Batay virus and Tyna virus, so as to solve the problem of lack of an effective detection method for Batay virus and Tyna virus

Method used

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  • Primer probe group and kit for combined detection of Batai virus and Taina virus based on dual fluorescence PCR method
  • Primer probe group and kit for combined detection of Batai virus and Taina virus based on dual fluorescence PCR method
  • Primer probe group and kit for combined detection of Batai virus and Taina virus based on dual fluorescence PCR method

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Experimental program
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Effect test

Embodiment 1

[0032] Design and synthesis of primers and probes for the dual detection kit (fluorescence PCR method) of Batay virus and Taina virus:

[0033] According to SEQ ID No.1-6, synthesize the specific primers of Batai virus, the Batai virus probe, the specific primers for Taina virus and the Taina virus probe, and label FAM at the 5' of Batay virus probe The fluorophore, the 5'-labeled HEX fluorescent group of the Tyna virus probe, and the 3' end of both are labeled with BHQ1, and the primer probes for Batay virus and Tyna virus are synthesized. The primers and probe sequences are as follows:

[0034] Batay virus:

[0035] Upstream primer: ATTAACTTTAAGCGTATCTA (SEQ ID No.1)

[0036] Downstream primer: GTATTAAATACAGTAACCTTC (SEQ ID No.2)

[0037] Probe: 5'FAM-TTAGTTATGACAACATTAGAATCTT-BHQ13' (SEQ ID No.5)

[0038] Tynavirus:

[0039] Upstream primer: TATGATAAATACTATGAACTAAC (SEQ ID No.3)

[0040] Downstream primer: AATCTATTGGAGCTAATATG (SEQ ID No.4)

[0041] Probe: 5'HEX-ATCTATT...

Embodiment 2

[0043] Preparation of detection mixture of Batay virus and Taina virus dual nucleic acid detection kit (fluorescent PCR method):

[0044] According to Batai virus upstream primer 0.5μl / test; downstream primer 0.5μl / test; probe 0.25μl / test; Taina virus upstream primer 0.5μl / test; downstream primer 0.5μl / test; probe 0.25μl / test; primer The concentration is 10μM, and the probe concentration is 10μM; PCR MIX 12.5μl / test; process water (ddH 2 O) Mix at 5 μl / test to obtain the nucleic acid fluorescent PCR detection mixture.

Embodiment 3

[0046] Sensitivity analysis of Batai virus and Taina virus dual nucleic acid detection kit (fluorescent PCR method):

[0047] 3.1 Sample preparation:

[0048] Take 1×10 9 Copies / ml of Batai virus plasmid (BATV-S1) (the BATV-S1 plasmid obtained by cloning the target amplified sequence into the PEGM-T vector by TA. The target amplified sequence is:

[0049] ATTAACTTTAAGCGTATCTACACCACTGGGCTTAGTTATGACAACATTAGAATCTTCTACATTAAAGGACGCGAGATTAAAACTAGTCTCGCAAAAAGAGTGAATGGGAGGTTACGCTTAACCTTGGGGGCTGGAAGGTTACTGTATTTAATAC (SEQ ID No. 7)) was divided into 5 parts, and 4 parts were diluted according to 10 times, 100 times, 1000 times, 10000 times to obtain BA0 TV 8 copies / ml), BATV-S3 (1×10 7 copies / ml), BATV-S4 (1×10 6 copies / ml), BATV-S5 (1×10 5 copies / ml), a total of 5 copies were obtained as test samples.

[0050] Take 1×10 9 Copies / ml Taina virus plasmid (TAHV-S1) (the TAHV-S1 plasmid obtained by cloning the target amplification sequence into the PEGM-T vector by TA. The target ampl...

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Abstract

The invention discloses a primer probe group for combined detection of Batai virus and Taina virus based on a dual fluorescence PCR method. The primer probe group contains specific primers and probesaiming at the Batai virus as well as primers and probes aiming at the Taina virus, wherein the specific primers aiming at the Batai virus include a forward primer and a reverse primer, the nucleotidesequence of the forward primer aiming at the Batai virus is represented by SEQ ID No.1, and the nucleotide sequence of the reverse primer aiming at the Batai virus is represented by SEQ ID No.2; and the specific primers aiming at the Taina virus include a forward primer and a reverse primer, the nucleotide sequence of the forward primer aiming at the Taina virus is represented by SEQ ID No.3, andthe nucleotide sequence of the reverse primer aiming at the Taina virus is represented by SEQ ID No.4. The primer probe group and the kit utilizing the primer probe group can be used for effectively detecting the Batai virus and the Taina virus, and the detection sensitivity can reach 1*10<3>copies / ml, so that the detection blanks of the Batai virus and the Taina virus in the prior art are effectively overcome, and the primer probe group and the kit have high industrial utilization values.

Description

technical field [0001] The present invention relates to a primer probe set and kit for joint detection of Batay virus and Tyna virus, in particular to a primer probe set and kit for joint detection of Batay virus and Tyna virus based on double fluorescent PCR method and how to use it. Background technique [0002] Batai virus (BATV) belongs to the order Bunyavirus, the family Peribunyavirus, and the genus Bunyavirus. It was first isolated from Culex mosquitoes in Malaysia in the 1950s, and then It is isolated from various media in many countries and regions such as Europe and Asia. Batay virus infection can cause influenza-like symptoms and occasionally viral encephalitis in humans, and the virus can cause severe infectious disease in ruminants, including abortion, premature birth, and genetic defects. Bartay virus is the most widely distributed virus in the Bunyaviridae family, and it is also the virus with the most diverse vectors. The main vectors include Anopheles macu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2521/107C12Q2537/143C12Q2563/107
Inventor 吴德孙九峰张鲍欢谈琦琪梁楚敏张欢张欣周惠琼宁丹
Owner 广东省公共卫生研究院
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