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RT-PCR quantitative detection method for hantavirus

A quantitative detection method, RT-PCR technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve the problem of long separation period, low success rate, and difficult separation of Hantavirus and other problems, to achieve the effect of simple operation, low cost and short reaction time

Inactive Publication Date: 2018-01-09
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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Problems solved by technology

Although the virus isolation method can provide direct evidence of virus infection, it is obviously not suitable for rapid clinical diagnosis because hantavirus is a difficult virus to isolate, and the isolation period is long and the success rate is not high.

Method used

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Embodiment 1

[0037] 1. Design of fluorescent quantitative PCR primers and probes:

[0038] A pair of PCR amplification primers and primer probes are designed according to the S gene sequence of Hantavirus 76-118 strain M14626, and the PCR amplification primers and primer probes are all synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and the Hanta Virus 76-118 strain M14626 was obtained directly from Qingdao Center for Disease Control and Prevention. The upstream primer of the PCR amplification primer is shown in SEQ ID No.1, the downstream primer is shown in SEQ ID No.2, and the primer probe is shown in SEQ ID No.3.

[0039] 2. Extract RNA from COS-1 subcultured cells infected with Hantavirus 76-118 strain:

[0040] Take the subcultured cells and put them in a centrifuge tube after centrifugation, add 300 μl Trizol, then add 100 μl chloroform, oscillate on the mixer for 5 seconds or invert for 15 times; centrifuge at 13000 rpm for 15 minutes, absorb the upper layer, add an equal ...

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Abstract

The invention provides a RT-PCR quantitative detection method for hantavirus. The method comprises the following steps: designing a PCR amplification primer and probe by an S gene sequence of hantavirus; with a COS-1 cell of hantavirus as a host cell, extracting RNA; performing PCR amplification on the S gene fragment of a cDNA product; connecting the PCR amplification product to a pMD18-T carrier; constructing recombinant plasmids and extracting the same; determining the copy number of the recombinant plasmids; diluting the same to obtain a plurality of standard articles of various concentrations; amplifying the plurality of standard articles by means of a real-time PCR program; detecting by fluorescence detection to obtain a Ct value; and establishing a standard curve by taking the Ct value and the concentration value of the standard articles as coordinates. The method provided by the invention tests hantavirus quantitatively and a method of sensitively and specifically detecting theviral load of hantavirus is established. The method has the advantages of simple operation, short reaction time, low cost and the like, and is in particularly suitable for clinical and scientific research.

Description

technical field [0001] The invention belongs to the technical field of virus detection, in particular to a RT-PCR quantitative detection method for hantavirus. Background technique [0002] my country is the most serious country for hemorrhagic fever with renal syndrome (HFRS), which is caused by Hantavirus infection. Clinical research has confirmed that "early discovery, early rest, early treatment, and nearest treatment" are the decisive factors for the prognosis of HFRS, and all treatment measures must be based on "early". Therefore, early diagnosis of patients is of great significance to the treatment of HFRS. Although traditional serological methods have strong specificity and high sensitivity, they still have certain limitations in the early diagnosis of HFRS. Although the virus isolation method can provide direct evidence of virus infection, it is obviously not suitable for rapid clinical diagnosis because hantavirus is a difficult virus to isolate, and the isolatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12Q1/686C12R1/93
Inventor 吴国才朱元祺赵辉
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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