Kit and special primer for detecting H1N1 influenza A virus and target sequence

An influenza virus and kit technology, applied in the field of influenza virus detection kits, can solve the problems of complicated operation steps and long reaction time, and achieve the effects of simple identification method, short detection time and high sensitivity

Inactive Publication Date: 2009-12-02
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity and specificity of the PCR method are lower than that of the ring-mediated isothermal amplification method, and the operation steps are cumbersome and the reaction time is long; while the real-time quantitative RT-PCR method requires an expensive real-time quantitative PCR instrument, and the results require computer analysis

Method used

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  • Kit and special primer for detecting H1N1 influenza A virus and target sequence
  • Kit and special primer for detecting H1N1 influenza A virus and target sequence
  • Kit and special primer for detecting H1N1 influenza A virus and target sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Design of target sequences and special primers for detection of type A H1N1 virus

[0036] (1) The target sequence designed to detect the H1N1 virus

[0037]According to the sequence of the type A H1N1 gene reported by Genebank, sequence number GI: 227977103, the entire influenza virus library was analyzed by bioinformatics, and the specific target sequence HA1 for the HA region of type A H1N1 nucleic acid was screened, which is located in the 301st HA gene. base to base 580. Its base sequence is shown in SEQ ID No.1.

[0038] (2) Specific primers designed to detect the target sequence of H1N1 virus

[0039] The method is as follows: according to the gene sequence of the HA segment gene of the influenza A H1N1 virus nucleic acid with the sequence number of GI: 227977103 in Genebank and the target sequence HA1 of the HA region specific for the H1N1 gene selected above, primer design and specificity analysis are integrated. The software tool BioSun designed 4...

Embodiment 2

[0044] Example 2 Preparation of the positive control HA1 RNA fragment of the kit of the present invention

[0045] (1) Construction of pMD-20T-HA1 plasmid

[0046] Take 1 ul of the recombinant expression plasmid pBluescript IISK(+)-HA1 purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd., use CaCl 2 Methods The competent cells DH5α were transformed, and the recombinant clones were screened out by using LB solid medium containing ampicillin resistance, and the plasmids were extracted. Using the extracted plasmid of pBluescript II SK(+)-HA1 containing HA1 as a template, with F2, that is, the base sequence of SEQ ID No. 4 and B2, that is, the base sequence of SEQ ID No. 5, as primers, PCR amplification of HA1 Fragment, the reaction volume is 20 μL:

[0047] pBluescript II SK(+)-HA1 plasmid (0.5 μg): 1 μL

[0048] F2 (5μM): 0.5μL

[0049] B2 (5μM): 0.5μL

[0050] dNTP (2.5mM): 1.6RNA

[0051] 10×PCR buffer: 2μL

[0052] Taq enzyme (5U / μL): 0.2μL

[00...

Embodiment 3

[0068] Embodiment 3 utilizes the method for detecting type A H1N1 virus with the kit of the present invention

[0069] (1), a special kit for the detection of type A H1N1 virus

[0070] The components of the kit for detecting 20 samples and their source or preparation method:

[0071] 40U / μL RNase inhibitor, purchased from Bao Bioengineering (Dalian) Co., Ltd., 1 tube, a total of 21 μL;

[0072] 8U / μL Bst DNA polymerase, purchased from Bailingke Biotechnology Company, 1 tube, a total of 21 μL;

[0073] 5U / μL AMV reverse transcriptase, purchased from Bao Bioengineering (Dalian) Co., Ltd., 1 tube, a total of 21 μL;

[0074] RNase free ddH 2 O, 1 tube, a total of 100 μL;

[0075] 500×SYBR Green I, purchased from Debilin Technology Co., Ltd., 1 tube, 42 μL in total;

[0076] 1.41×10 -21 M's positive template and 1.3 x 10 -20 The negative template of M, each one tube, is 25 μL; wherein, the preparation method of the positive template HA1 RNA is as in Example 2, and the negat...

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PUM

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Abstract

The invention discloses a special primer for detecting H1N1 influenza A virus, a kit comprising the primer for detecting H1N1 influenza A virus and a target sequence corresponding to the primer, and belongs to the field of quick diagnosis of pathogen genes. The base sequence of the target sequence corresponding to the special primer is shown as SEQ ID No.1; and the kit utilizes the technical principles of genetic reverse transcription and isothermal amplification to quickly detect H1N1 influenza A virus nucleic acid, and the detection result can be judged by naked eyes and also can be analyzed by using electrophoresis. The kit has the characteristics of quickness, sensitivity, specificity and convenient operation.

Description

technical field [0001] The invention relates to a kit for detecting influenza virus, in particular to a kit for detecting influenza A H1N1 virus, a special primer and a target sequence for the primer. Background technique [0002] Since mid-to-late April 2009, the H1N1 influenza epidemic originated in North America began to spread in many countries around the world. As of 14:00 on June 9, Beijing time, the World Health Organization confirmed that 74 countries and regions in the world have a total of 25,288 confirmed cases of Influenza A (H1N1), including 139 deaths. So far, a total of 101 confirmed cases of Influenza A (H1N1) have been reported in mainland China. Influenza A (H1N1) has caused a serious threat to human health, seriously endangering people's health and causing great economic losses. Rapid and accurate detection of influenza virus is the key to the prevention and control of influenza A H1N1. On the one hand, it can provide evidence and buy time for prevention...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 徐东刚李伍举蔡欣付文亮曹源邹民吉
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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