Nucleotide sequence, method and kit for detecting human influenza A virus

An influenza A virus, nucleotide sequence technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve problems such as difficult specific diagnosis, false positives, cross-reactions, etc.

Inactive Publication Date: 2012-11-21
CHINESE NAT HUMAN GENOME CENT AT SHANGHAI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods still have certain defects, such as the low sequence specificity of the primers and probes used, it is difficult to carry out specific diagnosis for the multiple mutations scattered in the genome of the current new type A (H1N1) influenza virus, and it is easy to compare with previous seasonal variations. Influenza A (H1N1) viruses cross-react or fail to amplify new strains of influenza A (H1N1) viruses with mutations in primer or probe positions, resulting in false positive or false negative results

Method used

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  • Nucleotide sequence, method and kit for detecting human influenza A virus
  • Nucleotide sequence, method and kit for detecting human influenza A virus
  • Nucleotide sequence, method and kit for detecting human influenza A virus

Examples

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Effect test

Embodiment 1

[0062] Example 1 Influenza A virus, seasonal influenza A virus and new type A H1N1 influenza virus typing detection method and kit

[0063] 1: material

[0064] 1.1 Virus samples:

[0065] 1 swine influenza virus sample (virus sample from: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences);

[0066] 1 sample of the new epidemic influenza A (H1N1) virus (virus sample from: Hong Kong Queen Mary Hospital);

[0067] 3 samples of seasonal influenza A virus: 1 seasonal influenza A H1, 1 seasonal influenza A H3, 1 seasonal influenza A H9 (virus samples from: Guangzhou Center for Disease Control and Prevention) ;

[0068] 1 highly pathogenic H5N1 avian influenza virus sample (virus sample from: Hong Kong Queen Mary Hospital);

[0069] 1 copy of influenza B virus (virus sample from: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences).

[0070] 2. Method

[0071] 2.1 Acquisition of total RNA of the virus strain

[0072]...

Embodiment 2

[0089] Example 2 Kit

[0090] The specification of this kit is for 20 people, and the composition of the kit is:

[0091]

[0092] Among them, RT-PCR reaction solution: 100 μl of 10× reverse transcription-PCR reaction solution, 40 μl of 10 mM dNTP, 200 μl of 5× reverse transcription enhancement solution, 10 μl of RNase inhibitor, 20 μl of 20 μM primers and probes (SEQ ID NO: 1 -12), to 800μl, mix well.

[0093] Mixed enzyme solution: 10 μl reverse transcriptase and 30 μl hot-start DNA polymerase.

[0094] The positive control substance is a solution containing plasmids pMD19A, pMD19H1 and pMD19RnaseP, the new type A H1N1 quantitative reference substance is a solution containing plasmid pMD19H1N1, and the negative control substance is the oral secretion of normal people.

Embodiment 3

[0095] The usage method of embodiment 3 kit:

[0096] 1. Reagent Preparation

[0097] 1.1 Thaw the reagent completely before opening the tube, mix well and centrifuge briefly

[0098] 1.2 Preparation: Calculate by taking 43 μl of RT-PCR reaction solution and 2 μl of mixed enzyme for each reaction, multiply by the number of reaction tubes required (it is recommended to add one more reaction volume as appropriate), add them to a total sterile centrifuge tube, Vortex to mix.

[0099] 1.3 Dispensing: Add 45 μl of the above-mentioned mixed reaction solution into each PCR reaction tube with a micro-sampler, and store at 4°C.

[0100] Note: It is best to prepare the reaction solution after the RNA extraction is completed.

[0101] 2. Sample RNA extraction: Use Qiagen's RNA kit to process the sample, extract and purify the total RNA, dissolve it in 50ul DEPC water, and follow the manufacturer's instructions for the entire operation.

[0102] 3. Adding samples: Add 5ul of the sampl...

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Abstract

The invention relates to a nucleotide sequence, a method and a kit for detecting human influenza A virus, which can detect human influenza A virus, influenza A(H1) virus and novel influenza A H1N1 virus by genotyping. A primer and a probe comprise any one of the nucleotide sequences as shown in the figures from SEQ ID NO:1 to SEQ ID NO:12. The invention can respectively detect the human influenza A virus, the influenza A(H1) virus and the novel influenza A H1N1 virus in the same fluorescence PCR reaction system, adopts interior labels to carry out quality control on the nucleic acid extraction and PCR reaction processes, eliminates false negative generation, enables the whole experimental detection process to be accurate and reliable, improves the early-stage relevance ratio, reduces the operating time of medical personnel, lowers the cost and lightens economical burden for patients.

Description

technical field [0001] The invention relates to a nucleotide sequence, method and kit for detecting human influenza A virus, in particular to a set of primer pairs and probes for detecting human influenza A virus by multiplex fluorescent PCR, and detection Method Kit. Background technique [0002] Influenza is a contagious disease caused by the influenza virus that spreads through the respiratory tract. Influenza virus belongs to the Orthomyxoviridae family and the genus Influenza virus. According to the different antigenic and genetic characteristics of the virus, influenza viruses are divided into three types: A, B, and C. Influenza A virus can be divided into many subtypes according to the different H and N antigens. H can be divided into 15 subtypes (H1-H15), and N has 9 subtypes (N1-N9). Among them, only H1, H3, and H9 mainly infect humans, and the natural hosts of many other subtypes are various birds and animals. In general, bird flu viruses do not infect animals o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64C12R1/93
Inventor 董辉熊慧金维荣李辉丁国徽谢立群陈嘉铮严安车小燕袁国勇赵国屏
Owner CHINESE NAT HUMAN GENOME CENT AT SHANGHAI
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