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Isothermal amplification detection kit for bird flu H5N1 virus and detecting method thereof

A constant temperature amplification detection and constant temperature amplification technology, which is applied in the field of in vitro diagnostic reagents, can solve the problems of expensive and false positives, and achieve the effects of fast reaction speed, simple steps, and reduced chances of amplification product contamination

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

[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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  • Isothermal amplification detection kit for bird flu H5N1 virus and detecting method thereof
  • Isothermal amplification detection kit for bird flu H5N1 virus and detecting method thereof
  • Isothermal amplification detection kit for bird flu H5N1 virus and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The composition and preparation of the kit

[0044] a) RNA extraction reagent: RNA extraction kit

[0045] 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:

[0046] The peripheral primers are:

[0047] The forward peripheral primer sequence is 5'-GGAGTTTCTTCTGGACAA-3' (SEQ ID NO1);

[0048]The reverse peripheral primer sequence is 5'-GTCGCAAGGACTAATCT-3' (SEQ ID NO2);

[0049] The sequences of the two probes are:

[0050] Forward 5' end Biotin labeled probe 5'-biotin-GAGTCCCCCTTTCTTGACAAT-3' (SEQ ID NO3);

[0051] Reverse 3' end fluorescein isothiocyanate FitC labeled probe 5'-GATAAACTCTAGTATGCCA-FitC-3' (SEQ ID NO4);

[0052] The amplification cross primers are:

[0053] Amplify reverse primer 5'-ATGGTGAGAGGGTGTATTCATTGCTCCAGAATATGC-3' (SE...

Embodiment 2

[0061] Concrete method for detecting avian influenza H5N1 virus nucleic acid with kit of the present invention

[0062] a) Extract RNA from the specimen to be tested with an RNA extraction kit.

[0063] 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 .

[0064] 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 avian influenza H5N1 virus nucleic acid, the detection line of the test strip is positive.

[0065] 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 compa...

Embodiment 3

[0067] The specificity of detecting avian influenza H5N1 virus with the kit of the present invention

[0068] 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

[0069] Table 1 Specific detection results of avian influenza H5N1 virus

[0070] serial number

name

Test results

1

A-H3N2

-

2

A-H5N1

-

3

Type A H9N7

-

4

Influenza A (H1N1)

-

5

Seasonal Influenza B

-

[0071] serial number

name

Test results

6

Avian influenza H5N1

+

7

Human Seasonal Influenza H1N1

-

[0072] Note: "-" means negative, "+" means positive

[0073] It can be seen from the test results in Table 1 that the detection of avian influenza H5N1 vir...

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Abstract

The invention relates to an isothermal amplification detection kit for bird flu H5N1 virus and a detecting method thereof. The kit comprises RNA extract, bird flu H5N1 virus nucleic acid isothermal amplification reaction liquid, bird flu H5N1 virus positive control and negative control. The detection kit has the advantages of high specificity, high sensitivity, high reaction speed, suitability for sample detection of both high flux and low flux and no need of complicated instrument in the whole reacting process, wherein only 2 hours are required from treating a single sample to finishing detection.

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 for avian influenza H5N1 virus and a detection method thereof. Background technique [0002] Human avian influenza, namely human avian influenza, is an acute respiratory infectious disease caused by certain subtypes of avian influenza A virus. In May 1997, a 3-year-old child in Hong Kong Special Administrative Region of China died of unexplained multiple organ failure. In August of the same year, it was identified as poultry by the Centers for Disease Control and Prevention of the United States, the World Health Organization (WHO), and the National Influenza Center in Rotterdam, the Netherlands. Human influenza caused by influenza A virus H5N1, this is the first time in the world that avian influenza A virus H5N1 has been confirmed to infect humans. [0003] The avian influenza caused by the avian influenza ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 章琪叶魏阎俊张琳韩晓辉吴嘉平张晓航王健孟成艳周娴成玉明简大钊张宏胡林吴汀滢王宏莹李波
Owner 上海国际旅行卫生保健中心
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