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RAA primer probe and detection method for detecting Hanning virus

A primer-probe and probe technology, applied in the field of virus detection, can solve the problems of high personnel requirements, expensive instruments, long detection time, etc., and achieve the effect of reducing detection time, lowering detection cost, and shortening detection time.

Pending Publication Date: 2022-05-13
SHANDONG BOSIYUAN BIOLOGICAL TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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 JUNV is a problem that those skilled in the art need to solve urgently

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0061] The method for RAA fluorescence method detection JUNV virus, comprises the steps:

[0062] Homogenize the tissue of the sample to be tested, extract nucleic acid according to the method of extracting RNA from 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;

[0063] Turn on the constant temperature fluorescent gene detector RAA-F1620 to preheat, and set the reaction parameters. The reaction parameters are set to 39°C, and the reaction time is 20 minutes;

[0064] 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;

[0065] Add 2.5 μL of Mg2+ to the cap of the reaction tube, fully mix 5 μL of the nucleic acid extraction solution obtained in step (1) ...

Embodiment approach

[0079] Step 1. Prepare reaction solution (prepared according to 10 reactions):

[0080] Draw 250 μL from the reaction buffer and add it to a pre-prepared 1.5mLEP tube, add 167 μL water, 12.6 μL primer, and 3.6 μL probe (the concentration of the primer is 10 μM, and the concentration of the probe is 10 μM), mix well, and mix well. Homogenized reaction solution.

[0081] Step 2, RAA fluorescence basic reaction reagent redissolution

[0082] Prepare 6 RAA fluorescent basic reaction reagents, draw 46.5 μL of the reaction buffer mixed in step 1 each time, and add them to the prepared 6 RAA fluorescent basic reaction reagent tubes, so that the lyophilized powder is fully dissolved and mixed to become RAA reaction system, and marked.

[0083] Step 3. Sample addition reaction

[0084] After adding 2.5 μL Mg2+ to the caps of the above 6 prepared RAA fluorescent basic reaction reagent test tubes, add 1 μL negative quality control substance and 1 μL each of standard working products 1...

Embodiment 3

[0121] Embodiment 3 actual sample detection

[0122] The sequences of primers, probes and negative quality controls are the same as in Example 1.

[0123] In the experiment, 8 clinical samples 1 to 8 were provided by Ningbo International Travel Health Care Center;

[0124] Sample extraction method:

[0125] Homogenize the tissue samples first, and then extract nucleic acid according to the method of extracting RNA from Tiangen’s commercial tissue; extract nucleic acid from serum and plasma by lysing, magnetic bead enrichment, washing, and elution; store at -20°C for later use;

[0126] Method of implementation

[0127] Step 1. Prepare reaction solution (prepared according to 10 reactions):

[0128] Draw 250 μL from the reaction buffer and add it to a pre-prepared 1.5mLEP tube, add 167 μL of water, 21 μL of primers, and 6 μL of probes (the concentration of primers is 10 μM, and the concentration of probes is 10 μM), mix well, and mix well. the subsequent reaction solution. ...

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Abstract

According to the primer, the probe and the detection method for detecting the JUNV by the fluorescent RT-RAA method, the detection of the JUNV can be completed within 20 minutes under the condition of 39 DEG C, and the primer, the probe and the detection method have the characteristics of rapidness, sensitivity, simplicity and convenience in operation and suitability 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 Jiuning virus. Background technique [0002] Junin virus (JUNV) can cause acute febrile disease and can cause Argentine hemorrhagic fever, which is a natural foci disease transmitted by rodents. Clinical features include fever, severe myalgia, hemorrhage, shock, neurological abnormalities, and leukopenia and thrombocytopenia. The incubation period is 6-14 days. The onset is slow. Most of them are men and young adults. Those who are seriously ill may suffer from coma and shock, and a few die within 48 to 72 hours. JUNV virus is an RNA virus with a diameter of 60-280nm and an average of 110-130nm. It is spherical, oblate or multi-type. There are 2 to 10 rod-shaped projections about 6 nm in length on the outer membrane, and 2 to 10 sand-like particles with a diameter of 20 to 25 nm in the virion. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2531/119C12Q2563/107Y02A50/30
Inventor 周冬根倪敏君
Owner SHANDONG BOSIYUAN BIOLOGICAL TECH CO LTD
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