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Kit for detecting dengue fever virus, and special-purpose amplification primer and probe for the same

A technology of dengue fever virus and kits, which is applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of cumbersome detection process, cumbersome operation, and long time consumption, and achieve high sensitivity, Strong specificity and easy operation

Inactive Publication Date: 2008-01-09
SHENZHEN INT TRAVEL HEALTHCARE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, the most primitive and classic method for dengue virus detection is the isolation and cultivation of specific viruses, but it is difficult to popularize in practice due to cumbersome operations, high technical difficulty and time-consuming, high requirements for experimental conditions, and serious biosafety problems application
Compared with virus isolation culture and gene detection method, ELISA method will produce certain false positive
Moreover, the PCR detection kits for dengue virus currently on the market can only detect dengue types I\II\III\IV separately, which takes a long time and the detection process is cumbersome.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, the design of the special-purpose primer and probe that detects dengue fever virus

[0018] According to the gene sequence analysis of dengue fever I-IV virus in NCBI database, the conserved sequences of Genbank numbers EF032590, NC_001474, DQ401690, and AF326573 were compared with ClustalW software to find out the same sequence in the conserved sequence region, and analyzed by NCBI-Blast and Other viruses and microorganisms have no sequence homology, and probes were designed in this region.

[0019] The special primer is composed of a forward primer and a reverse primer, the base sequence of the forward primer is shown in SEQ IN NO:1, and the base sequence of the reverse primer is shown in SEQ IN NO:2. The base sequence of the dedicated probe is shown in SEQIN NO:3.

Embodiment 2

[0020] Embodiment 2, use the kit of the present invention to carry out fluorescence quantitative detection on the sample to be tested

[0021] 1. Preparation of reaction solution

[0022] Each reaction solution is 18 μl, and each reaction solution contains the following components: 10 μl Probe RT-PCR Master Mix, 0.2 μl Probe RT-PCR Buffer, 1 μl upstream primer (10 pmol / μl), 1 μl 1.0 μM downstream primer (10 pmol / μl), 0.25 μl probe (10 pmol / μl), 5.55 μl of DEPC water.

[0023] 2. Sample collection

[0024] Serum: Use a disposable sterile syringe to extract 2 ml of venous blood from the subject, inject it into a sterile dry glass tube, and place it at room temperature (22-25°C) for 30-60 minutes. Centrifuge at 1500rpm for 5 minutes; absorb the upper serum and transfer it to a 1.5ml sterilized centrifuge tube for later use.

[0025] Plasma: Use a disposable sterile syringe to extract 2 ml of venous blood from the subject, inject it into a glass tube containing EDTA (disodium ...

Embodiment 3

[0056] Embodiment 3, determination of kit sensitivity and linear range of the present invention

[0057] 1. Determination of the sensitivity of the kit of the present invention

[0058] For the RNA prepared in step 4, after 10-fold dilution, the minimum concentration is 1.0×10IU / ml, detected in step 5, 1.0×10 3 Fluorescent signals can be detected above the concentration of IU / ml, so it is determined that the sensitivity of this kit to the processed samples is 1.0×10 3 IU / ml.

[0059] 2. Determination of the linear range of the kit of the present invention

[0060] The concentration of RNA is 1.0×10~1.0×10 10 IU / ml standard was tested and found that the concentration was less than 1.0×10 3 IU / ml and greater than 1.0×10 8 The growth curve of IU / ml is not an S-shaped curve, and is not within the linear range of detection. It can be seen that the linear range of the kit of the present invention is 1.0×10 3 ~1.0×10 8 IU / ml.

[0061] (1) If the growth curve does not present ...

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PUM

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Abstract

A kit and special alkali inducers for amplification and probes can be used to check out dengue fever virus. The inducers consist of a couple of opposite directions. One is of sequence shown in No.1, another is of sequence shown in No.2, and probes shown in No.3. The process has advantages, e.g. quick action, simple, high sensitivity and specification in detection of dengue fever virus I, II, III and IV.

Description

technical field [0001] The invention relates to a kit for detecting viruses and special amplification primers and probes thereof, in particular to a kit for detecting dengue virus (DV) and special amplification primers and probes thereof. Background technique [0002] Dengue fever is an acute arbovirus infectious disease with the widest distribution and the largest number of cases in the world, which is prevalent in tropical and subtropical regions. Dengue fever is caused by dengue virus, which is divided into 4 types, namely dengue I\II\III\IV, which are transmitted by Aedes mosquitoes. According to WHO statistics, 50 million people are infected with dengue fever every year, of which about 500,000 are infected with dengue hemorrhagic fever, more than 20,000 people die, and 2.5 billion people are threatened. Dengue fever has become the largest public health problem in the world. [0003] There have been dengue fever epidemics caused by dengue virus types I\II\III\IV in Chin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCY02A50/30
Inventor 朱玉兰吴兵王佃鹏徐云庆高朝贤金玉娟黄宗炎
Owner SHENZHEN INT TRAVEL HEALTHCARE CENT
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