Primer probe set and detection method for detecting severe fever with thrombocytopeniasyndrome virus by real-time fluorescence RNA RT-RPA (reverse transcription-recombinase polymerase amplification)

A real-time fluorescence and primer-probe technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as unsuitable for on-site emergency detection tasks, complex operation, large volume, etc., to achieve efficient real-time Fluorescent RNA is rapidly amplified at constant temperature, easy to operate, and has broad application prospects

Inactive Publication Date: 2021-02-09
中国疾病预防控制中心病毒病预防控制所
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0003] Traditional PCR technology relies on temperature changes to achieve amplification reactions such as denaturation, renaturation, and extension. The completion of amplification technology requires sophisticated and complex large-scale instrument control, such as temperature control and adjustment instruments and complex sample processing steps. These equipment are large in size, The price is expensive and the operation is complicated, so it is not suitable for on-site emergency detection tasks during the outbreak

Method used

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  • Primer probe set and detection method for detecting severe fever with thrombocytopeniasyndrome virus by real-time fluorescence RNA RT-RPA (reverse transcription-recombinase polymerase amplification)
  • Primer probe set and detection method for detecting severe fever with thrombocytopeniasyndrome virus by real-time fluorescence RNA RT-RPA (reverse transcription-recombinase polymerase amplification)
  • Primer probe set and detection method for detecting severe fever with thrombocytopeniasyndrome virus by real-time fluorescence RNA RT-RPA (reverse transcription-recombinase polymerase amplification)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of In vitro Transcribed RNA of Fever with Thrombocytopenia Syndrome Virus NP Gene

[0044] In this example, the in vitro transcribed RNA of the NP gene of fever with thrombocytopenia syndrome virus was prepared by using the in vitro transcription method, so as to provide a positive RNA template for subsequent detection experiments.

[0045] 1. PCR amplification of NP gene containing T7 promoter

[0046] The PCR method is used to introduce the T7 promoter into the upstream of the positive-strand reading frame and the downstream of the negative-strand reading frame of the fever with thrombocytopenia syndrome virus, so as to obtain RNA consistent with the viral genome sequence after in vitro transcription. Using the cDNA synthesized by reverse transcription of viral RNA as a template, use the FastStart High Fidelity PCR system to prepare the following PCR reaction system:

[0047]

[0048] Upstream primer (SEQ ID NO.5): 5'-TTGTGAGCGGATAACAATTCCC-3...

Embodiment 2

[0077] Example 2 Design of primers and probes suitable for real-time fluorescent RNA constant temperature rapid amplification

[0078] Through the sequence comparison of the NP gene of fever with thrombocytopenia syndrome virus, the conserved region was found, and primers and probes suitable for real-time fluorescent RNA constant temperature and rapid amplification technology were designed for the conserved region.

[0079] The general principles of primer and probe design for RNA isothermal rapid amplification are as follows:

[0080] (1) The length of the probe is generally between 46-52.

[0081] (2) The probe generally includes a fluorescent group, a quencher group, tetrahydrofuran (THF) and a block at the 3' end.

[0082] (3) The 5' end of THF needs to have at least 30 bases, and the 3' end generally needs to have 15 bases. THF is located between the fluorescent group and the quencher group, generally between the fluorescent group and the quencher group 2-4 bases apart....

Embodiment 3

[0121] Example 3 Method for detecting fever with thrombocytopenia syndrome virus by constant temperature and rapid amplification of real-time fluorescent RNA

[0122] Using the primers and probes shown in Table 7, use the RNA Constant Temperature Rapid Amplification Kit (Anpu Future, fluorescent type) to perform real-time fluorescent RNA constant temperature rapid amplification. The specific method is as follows:

[0123] Add 29.4 μl of rehydration buffer (Buffer A), 2 μl of upstream and downstream primers (10 μM), 0.6 μl of probe (10 μM), 0.5 μl of 40 U / μl RNase inhibitor and ddH 2 O 7.5 μl was mixed to obtain 42 μl of working buffer, and then 5 μl of the sample RNA to be tested was added as a template to mix with the above working buffer, and added to the enzyme amplification eight-tube tube equipped with a dry powdered enzyme system, and the Dissolve and mix well, and finally add 3 μl magnesium acetate solution (Buffer B) to the cap of the eight-tube tube, cover the cap car...

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Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a primer probe set and a detection method for detecting severe fever with thrombocytopeniasyndrome virus by real-time fluorescence RNA RT-RPA. The invention provides the primer probe set suitable for real-time fluorescence RNA RT-RPA for the severe fever with thrombocytopeniasyndrome virus, the nucleotide sequence of the primer probe set is shown as SEQ ID NO.2-4, and the primer probe set has high specificity and sensitivity. The real-time fluorescence RNA RT-RPA detection method provided by the inventioncan realize specific, sensitive, accurate and rapid detection of the severe fever with thrombocytopeniasyndrome virus, has the advantages of short detection time, simple operation, non-reliance on thermal cycle equipment and the like, can meet the requirements of field detection, and has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection, in particular to a target sequence for detecting fever with thrombocytopenia syndrome virus, a specific primer probe set, a kit, and the detection of fever with thrombocytopenia syndrome using real-time fluorescent RNA constant temperature rapid amplification technology way of the virus. Background technique [0002] Severe fever with thrombocytopenia syndrome (SFTS) is a serious infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV) infection, the main clinical symptoms are fever, Thrombocytopenia and leukopenia, gastrointestinal symptoms, abnormal liver and kidney function, etc. FTSV is mainly transmitted through tick bites, but direct contact with blood and body fluids of cases can also cause human-to-human transmission. Therefore, early diagnosis of FTSV is the key to epidemic prevention and control. Viral nucleic acid detection technology can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2561/113C12Q2563/107
Inventor 李阿茜梁米芳芜为黄晓霞李川李德新李建东
Owner 中国疾病预防控制中心病毒病预防控制所
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