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

A technology of getta virus, primer probe, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc.

Active Publication Date: 2018-12-07
广东省疾病预防控制中心 +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 for joint detection of Sindbis virus and Geta virus ba

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Design and synthesis of detection primer probes for Sindbis virus and Geta virus double detection kit (fluorescent PCR method):

[0033] Synthesize Sindbis virus-specific primers, Sindbis virus probes, specific primers for Geta virus and Geta virus probes according to SEQ ID No.1-6, and label FAM at the 5' of Sindbis virus probes The fluorescent group, the 5'-labeled HEX fluorescent group of the Geta virus probe, and the 3' end of both are labeled with BHQ1, and the primer probes for Sindbis virus and Geta virus are synthesized. The primers and probe sequences are as follows:

[0034] Sindbis virus:

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

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

[0037] Probe: 5' fluorescent reporter group-CCAAGACATTCTACAAGTATATCTC-BHQ13' (SEQ ID No.5)

[0038] Geta virus:

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

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

[0041] ...

Embodiment 2

[0043] Sindbis virus and Geta virus double nucleic acid detection kit (fluorescent PCR method) detection mixture preparation:

[0044] According to Sindbis virus upstream primer 0.5μl / test; downstream primer 0.5μl / test; probe 0.25μl / test; Geita virus upstream primer 0.5μl / test; downstream primer 0.5μl / test; probe 0.25μl / test; The concentration is 10μM, 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 Sindbis Virus and Geta Virus Dual Nucleic Acid Detection Kit (Fluorescent PCR Method):

[0047] 3.1 Sample preparation:

[0048] Take 1×10 9 Copies / ml Sindbis virus plasmid (SINV-S1) (cloning the target amplification sequence to the PEGM-T vector by TA to obtain the SINV-S1 plasmid. The target amplification sequence is:

[0049] CTAAAGGTACATTTCAAATCACCCTGAAAAAGACATATGCACCAAGACATTCTACAAGTATATCTCCCGGCGTTGCACACAGCCAGTTACAGCTATTGTATCGACACTGCATTACGATGGAAAGATGAAAACCACGAACCCGTGCAAGAAGAACATTGAAATCGATAT (SEQ ID No. 7)) was divided into 5 parts, and 4 parts were obtained by 10 times, 100 times, 1000 times, 1000 times, 100, 8 copies / ml), SINV-S3 (1×10 7 copies / ml), SINV-S4 (1×10 6 copies / ml), SINV-S5 (1×10 5 copies / ml), a total of 5 copies were obtained as test samples.

[0050] Take 1×10 9 Geta virus plasmid (GETA-S1) of copies / ml (the GETA-S1 plasmid obtained by cloning the target amplified sequence into the PEGM-T vector by TA. The target amplified...

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Abstract

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

Description

technical field [0001] The invention relates to a primer probe set and a kit for joint detection of Sindbis virus and Geta virus, in particular to a primer probe set and kit for joint detection of Sindbis virus and Geta virus based on a double fluorescent PCR method and how to use it. Background technique [0002] Sindbis virus (SINV) belongs to the Togaviridae (Tongaviridae) Alphavirus (Alphavirus), can be transmitted through mosquito bites to cause zoonotic Sindbis virus disease, the virus in 1952 in the Nile Delta Sindbis area of ​​Egypt Isolated from Culex mosquito. Since then, the virus has been isolated again from mosquito specimens in India, South Africa, Australia, the Middle East, Russia, Sweden and other places. Infected by Sindbis virus, in addition to causing clinical symptoms such as fever, rash and arthralgia, it can also develop into a chronic disease, which is very harmful to health. Since my country isolated a strain of Sindbis virus from the serum of fev...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2537/143C12Q2563/107C12Q2521/107
Inventor 孙九峰张鲍欢张欣梁楚敏谈琦琪张欢周惠琼宁丹吴德
Owner 广东省疾病预防控制中心
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