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Isothermal amplification assay kit for human seasonal influenza H1N1 and detection method thereof

A technology for constant temperature amplification detection and seasonal influenza, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of expensive, false positives, etc., achieve fast response, simple steps, and reduce amplification products Pollution Chance Effect

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 assay kit for human seasonal influenza H1N1 and detection method thereof
  • Isothermal amplification assay kit for human seasonal influenza H1N1 and detection method thereof
  • Isothermal amplification assay kit for human seasonal influenza H1N1 and detection 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'-GAATCAACTACTACTGGACT-3' (SEQ ID NO1);

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

[0049] The sequences of the two probes are:

[0050] The sequences of the two probes are:

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

[0052] Reverse 3' end fluorescein isothiocyanate FitC labeled probe 5'-GGAGCTATAAACAGCAGTCT-FitC-3' (SEQID NO4);

[0053] The amplification cross primers are:

[0054] Amplify reverse pri...

Embodiment 2

[0062] Specific method for detecting human seasonal influenza H1N1 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 human seasonal influenza H1N1 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...

Embodiment 3

[0068] The specificity of detection of human seasonal influenza H1N1 by using the kit of the present invention

[0069] According to the method of Example 2, human seasonal influenza H3N2, human seasonal influenza H5N1, human seasonal influenza 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 Specific detection results of human seasonal influenza H1N1

[0071] serial number

name

Test results

1

Human seasonal influenza H3N2

-

2

Human seasonal influenza H5N1

-

3

Human Seasonal Influenza H9N7

-

[0072] serial number

name

Test results

4

Influenza A (H1N1)

-

5

Seasonal Influenza B

-

6

Avian influenza H5N1

-

7

Human Seasonal Influenza H1N1

+

[0073] Note: "-" means negative, "+" mea...

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Abstract

The invention relates to an isothermal amplification assay kit for human seasonal influenza H1N1 and a detection method thereof. The assay kit comprises RNA extract, human seasonal influenza virus H1N1 nucleic acid isothermal amplification reaction liquid, and human seasonal influenza virus H1N1 positive control and negative control. The assay kit has the advantages of high specificity, high sensitivity, and high response speed. Only 2 hours are needed from processing to detection of a single sample; the assay kit can meet high-throughput and low-throughput sample detection simultaneously; and the whole reaction process does not need any complex instrument.

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 human seasonal influenza H1N1 and a detection method thereof. Background technique [0002] Seasonal influenza is caused by influenza viruses that have existed in the past. The epidemic is seasonal, with frequent outbreaks in winter and spring, and the population has certain immunity. The genotypes of influenza viruses that cause seasonal influenza mainly include H1N1, H3N2, and H5N1, among which influenza caused by H1N1 influenza virus accounts for the vast majority. [0003] Seasonal flu can cause symptoms such as fever, cough, sore throat, muscle pain, headache, chills, and fatigue. Seasonal influenza mainly affects children, the elderly, and some chronically ill people with weak constitutions. About 90% of severe cases and deaths occur in the elderly over 65 years old. Influenza vaccination is an ef...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 阎俊章琪叶魏韩晓辉张晓航王健周娴李克胜周心简大钊吴嘉平胡林徐高连石坚钟华燕
Owner 上海国际旅行卫生保健中心
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