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Constant-temperature amplification detection kit for dengue viruses and detection method

A technology of constant temperature amplification detection and dengue fever virus, which is applied in the field of qualitative detection of dengue fever virus, can solve problems such as expensive and false positives, and achieve the effects of fast reaction speed, simple steps and high sensitivity

Inactive Publication Date: 2014-03-05
狄飚 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although FQ-PCR has the advantages of simplicity, speed and sensitivity, its detection requires expensive instruments, and it is easy to cause false positives and other problems

Method used

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  • Constant-temperature amplification detection kit for dengue viruses and detection method
  • Constant-temperature amplification detection kit for dengue viruses and detection method
  • Constant-temperature amplification detection kit for dengue viruses and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Composition and preparation of embodiment 1 kit of the present invention

[0049] a) RNA extraction reagent: Germany QIAGEN RNA extraction kit (Qia-14162)

[0050]b) Reaction solution: two peripheral primers (0.05 μmol), two probes (0.5 μmol), and two cross primers (0.5 μmol), 1×Thermol buffer, MgSO 4 (6mmol), dNTPs solution (0.4mmol), 1xRNA secure, Bst DNA polymerase (8U), AMV reverse transcriptase (1U) and DEPC water, the total reaction volume is 16μl. in:

[0051] The peripheral primers are respectively:

[0052] Forward peripheral primer: 5'-ACTATGCTGCCTGTAGCTCC-3';

[0053] Reverse peripheral primer: 5'-CTGGAATGATGCTGAGGAGAC-3'.

[0054] The sequences of the two probes are respectively:

[0055] Forward 5' end Biotin labeled probe 5'-Biotin-CACTACGCCATGCGTACAGC-3';

[0056] Reverse 3' end fluorescein isothiocyanate (Fitc) labeled probe 5'-AGCGTCAATATGCTGTTTTTTG-Fitc-3'.

[0057] The amplification cross primers are respectively:

[0058] Reverse primer 5'-GG...

Embodiment 2

[0065] Embodiment 2 detects the concrete method of dengue fever virus nucleic acid with kit of the present invention

[0066] a) extracting RNA from the specimen to be tested with an RNA extraction kit;

[0067] b) Take the sample RNA as a template and add it to the PCR tube containing the reaction solution, and carry out the amplification reaction at 60°C for 90 minutes, wherein the sample RNA is 4 μl, and the reaction solution is 16 μl; a positive control template and a negative control template are respectively added to the control PCR tube;

[0068] c) Put the reacted PCR tube into the anti-pollution nucleic acid detection device for detection, and interpret the result after 15 minutes. When the sample contains dengue virus nucleic acid, the detection line of the test strip is positive;

[0069] The experiment was repeated 3 times, and there was no significant difference in the test results, indicating that the test results of different batches of this kit are comparable ...

Embodiment 3

[0070] Embodiment 3 kit of the present invention detects the specificity of dengue fever virus

[0071] According to the method of Example 2, influenza A H3N2, influenza A H5N1, seasonal influenza B, dengue virus, influenza A H9N7, influenza A H1N1, and human seasonal influenza H1N1 were simultaneously detected. Its detection result is as follows table 1 ( figure 2 Show):

[0072] serial number

name

Test results

1

A-H3N2

-

2

A-H5N1

-

3

Type A H9N7

-

4

Influenza A (H1N1)

-

5

Seasonal Influenza B

-

6

dengue virus

+

7

Human Seasonal Influenza H1N1

-

[0073] Table 1: Specific detection results of dengue virus

[0074] Note: "-" means negative, "+" means positive

[0075] It can be seen from the test results in Table 1 that the detection of dengue virus nucleic acid by the kit of the present invention has good specificity and strong specificity.

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Abstract

The invention provides a constant-temperature amplification detection kit for detecting dengue nucleic acids accurately, sensitively and rapidly, and a detection method. The detection kit is suitable for qualitative detection of dengue viruses (I, II, III and VI types). The kit comprises a constant-temperature amplification reaction solution, a positive control and a negative control. First, extracted dengue virus RNAs are subjected to amplification for 90min at a constant temperature of 60 DEG C through a cross primer constant-temperature amplification target nucleotide sequence method. Second, the products after amplification are hybridized with two probes with Biotin and fluorescein isothiocyanate (Fitc) labellings respectively in the constant-temperature amplification reaction solution, and the results are shown on nucleic acid test strips. Finally, detection reports are obtained through contrastive analysis with the positive and negative controls. The kit is advantaged by good specificity, high sensitivity and good repeatability. The kit can finish rapid detection of samples within 2h without complex apparatuses, and can meet detection requirements of high throughput and low throughput at the same time.

Description

technical field [0001] The invention relates to a rapid detection technology for dengue virus nucleic acid, which is suitable for qualitative detection of dengue virus (types I, II, III and VI). Background technique [0002] Dengue fever is an infectious disease caused by dengue fever virus, which is first bitten by Aedes albopictus and Aedes aegypti belonging to Aedes mosquito (also known as Aedes mosquito, Aedes mosquito) After the patient becomes a "vector mosquito", other healthy people may be infected by the bite of this vector mosquito. Symptoms of extreme fatigue and depression may occur. Occasionally, patients may develop dengue hemorrhagic fever, further bleeding, shock, and even death. Complications from dengue fever are often the main cause of death for patients. Generally speaking, dengue fever is mainly distributed in tropical and subtropical regions. [0003] Dengue virus is widely distributed between 25 degrees north latitude and 25 degrees south latitude. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
CPCC12Q1/6844C12Q1/70C12Q2531/119Y02A50/30
Inventor 狄飚白志军蒋力云
Owner 狄飚
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