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RAA primer probe for detecting Talavirus and detection method

A technology of primer probe and detection map, applied in the field of RAA primer probe and detection of Tula virus detection, can solve the problems of high personnel requirements, expensive instruments, long detection time, etc., to reduce detection time, reduce detection cost, The effect of short detection time

Pending Publication Date: 2022-04-12
SHANDONG BOSIYUAN BIOLOGICAL TECH CO LTD +1
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AI Technical Summary

Problems solved by technology

[0004] In conventional virus detection methods, virus isolation and identification, and serum neutralizing antibody tests are difficult to be widely used, and are not suitable for large-scale epidemiological investigations or on-site rapid diagnosis
However, the enzyme-linked immunosorbent assay (ELISA) has poor specificity, cross-reactivity, and false positives.
Although the fluorescent quantitative PCR method has high specificity, sensitivity and good repeatability, it still has the problems of expensive and huge equipment, high requirements for experimental sites and personnel, it is difficult to apply to large-scale on-site screening, and the detection time is too long. It takes 1 to 2 hours
All the above-mentioned methods are not suitable for on-site use at ports, and these methods require skilled personnel and well-equipped laboratories; , sensitive, easy to operate, and applicable to the fluorescence RT-RAA method for on-site rapid detection of TULV is an urgent problem to be solved by those skilled in the art

Method used

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  • RAA primer probe for detecting Talavirus and detection method
  • RAA primer probe for detecting Talavirus and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0055] The method for RAA fluorescence method detection JUNV virus, comprises the steps:

[0056] (1) Homogenize the tissue of the sample to be tested, extract the nucleic acid according to the method of extracting RNA from the tissue, and store it at -20°C for later use; if the sample is whole blood, serum, or plasma, use steps such as lysis, magnetic bead enrichment, washing, and elution to extract nucleic acid;

[0057] (2) Turn on the constant temperature fluorescent gene detector RAA-F1620 to preheat, set the reaction parameters, the reaction parameters are set to 39 ° C, and the reaction time: 20min;

[0058] (3) Add 12.7 μL of water to 25 μL of reaction buffer, 2.1 μL of upstream and downstream primers and 0.6 μL of probe at a concentration of 10 μM, mix well, add to RAA fluorescent basic reaction reagent and mix to obtain a reaction master mix;

[0059] (4) Add 2.5 μL of Mg to the cap of the reaction tube 2+ , fully mixing 5 μL of the nucleic acid extraction solution...

Embodiment 3

[0114] Embodiment 3 actual sample detection

[0115] (1) The sequences of primers, probes and negative quality controls are the same as in Example 1.

[0116] (2) Eight clinical samples 1 to 8 in the experiment were provided by Ningbo International Travel Health Care Center;

[0117] (3) Sample extraction method:

[0118] Homogenize tissue samples first, and then extract nucleic acid according to the method of extracting RNA from Tiangen commercialized tissue; extract nucleic acid from serum and plasma by lysing, magnetic bead enrichment, washing, and elution; store at -20°C for later use;

[0119] (4) Implementation method

[0120] Step 1. Prepare reaction solution (prepared according to 10 reactions):

[0121] Draw 250 μL from the reaction buffer and add it to a 1.5mLEP tube prepared in advance, add 167 μL of water, 21 μL of primers, and 6 μL of probes (the concentration of the primers is 10 μM, and the concentration of the probes is 10 μM), mix well, and mix well. Homog...

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Abstract

According to the RAA primer probe for detecting the Tara virus and the detection method, JUNV detection can be completed within 20 min at the temperature of 39 DEG C, and the RAA primer probe has the advantages of being rapid, sensitive, easy and convenient to operate and suitable for on-site rapid detection.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a RAA primer probe and a detection method for detecting Tula virus. Background technique [0002] Tula virus (TULV) is a member of the genus Hantavirus in the family Hantaviridae, which was isolated from the field mouse (micotus arvalis). TULV virus is a widely distributed virus in different parts of Eurasia. The common vole is its host, but it is also found in other voles. Clinical manifestations of TULV viral pathogenicity are very rare. In 2003, a case of hantavirus disease associated with TULV virus was diagnosed by serological and molecular epidemiological methods. So far, direct molecular evidence of TULV virus infection has been found in only 2 cases, one in an immunocompromised patient with severe hantavirus disease and the other in an immunocompetent patient with mild hantavirus disease. The ability of TULV virus to cause hantavirus disease even in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
Inventor 周冬根倪敏君
Owner SHANDONG BOSIYUAN BIOLOGICAL TECH CO LTD
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