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Constant-temperature amplification detection kit of A(H1N1) virus and detection method thereof

A technology of constant temperature amplification detection and constant temperature amplification, which is applied in the field of constant temperature amplification detection kits for type A H1N1 virus, can solve the problems of high cost and false positives, and achieve the effects of fast reaction speed, simple steps and high sensitivity

Inactive Publication Date: 2010-10-20
上海国际旅行卫生保健中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 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 of A(H1N1) virus and detection method thereof
  • Constant-temperature amplification detection kit of A(H1N1) virus and detection method thereof
  • Constant-temperature amplification detection kit of A(H1N1) virus and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The composition and preparation of the kit

[0045] a) RNA extraction reagent: RNA extraction kit

[0046] 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), Bst DNA polymerase (10U) and sterile double distilled water, the total reaction volume is 16μl. in:

[0047] The peripheral primers are:

[0048] The forward peripheral primer sequence is 5'-AATAACATTCGAAGCAACTGG-3' (SEQ ID NO1);

[0049] The reverse peripheral primer sequence is 5'-AAATAGGCCTCTAGATTGAAT-3' (SEQ ID NO2);

[0050] The sequences of the two probes are:

[0051] Forward 5' terminal Biotin labeled probe 5'-biotin-ATAATACCAGATCCAGCATT-3' (SEQ ID NO3);

[0052] Reverse 3' end fluorescein isothiocyanate FitC labeled probe 5'-AAAAGCACAAAATTGAGAC-FitC-3' (SEQ ID NO4);

[0053] The amplification cross primers are:

[0054] Amplify reverse primer 5'-GATATGCATTCGCAATGGAATTCCTCAATCCTG...

Embodiment 2

[0062] The concrete method that detects type A H1N1 virus nucleic acid with kit of the present invention

[0063] a) Extract RNA from the specimen to be tested with an RNA extraction kit.

[0064] 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, including 4 μl of the sample RNA and 16 μl of the reaction solution; add the positive control template and the negative control template respectively to the control PCR tube .

[0065] c) Put the reacted PCR tube into the nucleic acid anti-pollution detection device for detection, and interpret the result after 15 minutes. When the sample contains influenza A (H1N1) virus nucleic acid, the detection line of the test strip is positive.

[0066] 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 an...

Embodiment 3

[0068] Using the kit of the present invention to detect the specificity of type A H1N1 virus

[0069] According to the method of Example 2, influenza A H3N2, influenza A H5N1, influenza A H9N7, influenza A H1N1, seasonal influenza B, avian influenza H5N1, and human seasonal influenza H1N1 were detected. The results are shown in Table 1, see figure 1 .

[0070] Table 1 Specificity detection results of type A H1N1 virus

[0071] serial number

name

Test results

1

A-H3N2

-

2

A-H5N1

-

[0072] serial number

name

Test results

3

Type A H9N7

-

4

Influenza A (H1N1)

+

5

Seasonal Influenza B

-

6

Avian influenza H5N1

-

7

Human Seasonal Influenza H1N1

-

[0073] Note: "-" means negative, "+" means positive

[0074] As can be seen from the test results in Table 1, the detection of the A / H1N1 virus nucleic acid by the kit o...

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PUM

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Abstract

The invention relates to a constant-temperature amplification detection kit of an A(H1N1) virus and a detection method thereof. The kit comprises an RNA (Ribonucleic Acid) extracting solution, an A(H1N1) virus nucleic acid constant-temperature amplification reaction solution, A(H1N1) virus positive control and A(H1N1) virus negative control. The detection kit has high specificity, high sensitivity and high reaction speed, i.e. only two hours are needed from sample processing to detection finishing of a single sample; the sample detection of high flux and low flux can be simultaneously satisfied, and complicated instruments are not needed in the whole reaction process.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnostic reagents, in particular to a constant temperature amplification detection kit and a detection method for influenza A (H1N1) virus. Background technique [0002] When the H1N1 flu broke out in 2009, the World Health Organization initially used the name "swine flu". Now, WHO has begun to use "A (H1N1) influenza" instead of "swine flu" to refer to the current epidemic situation, and the announcement of the Ministry of Health of my country refers to this disease as "Influenza A (H1N1)". Influenza A (H1N1) virus is a type A influenza virus, carrying H1N1 subtype swine influenza virus strains, including ribonucleic acid gene fragments of three influenza viruses: avian influenza, swine influenza and human influenza, as well as Asian swine influenza and African swine Influenza virus characteristics. [0003] The clinical manifestations of influenza A H1N1 generally have an incubation period ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 叶魏阎俊简大钊王健陆晔张晓航张琳周娴孟成艳李克胜张宏尤其敏尤见徐高连王宏莹
Owner 上海国际旅行卫生保健中心
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