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Preparation method and application of gene chip for determination and drug resistance detection of influenza A virus

A type of influenza A virus, gene chip technology, applied in the field of gene chip detection, can solve the problems of long time, no specificity and sensitivity, etc.

Inactive Publication Date: 2012-05-02
深圳市普瑞康生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lu briefly reported that a chip can discriminate influenza A H1N1 and seasonal influenza virus. The virus RNA product is amplified by a two-step method and hybridized with the chip, which takes a long time and there is no experimental data on the specificity and sensitivity of this method.

Method used

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  • Preparation method and application of gene chip for determination and drug resistance detection of influenza A virus
  • Preparation method and application of gene chip for determination and drug resistance detection of influenza A virus
  • Preparation method and application of gene chip for determination and drug resistance detection of influenza A virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Development of Influenza A Virus Nucleic Acid Screening and Drug Resistance Detection Gene Chip

[0048] 1. General primer design and screening

[0049] In order to achieve simultaneous universal amplification of 4 subtypes of N1 influenza virus and 1 H3N2 influenza virus, 2 pairs of primers were used to amplify 4 N1 subtype viruses, and 1 pair of primers was used to amplify H3N2. The primer sequences and corresponding target See Table 1 for virus information. The combination of NF11 / NF12 and NR21 / NF22 amplified four N1 subtypes, the amplified fragment was 452bp, including the drug resistance mutation site H274Y; NF5 and NR5 amplified H3N2, and the amplified fragment was 281bp, including the drug resistance mutation site Point E119V. The agarose electrophoresis results of the amplified products are shown in the appendix figure 1 .

[0050] 2. Screening of specific probes

[0051] The screening of typing probes first excludes the non-specific crossover be...

Embodiment 2

[0059] Example 2: Determination of Gene Chip Positive Judgment Criteria

[0060] The cutoff value is a criterion for judging whether the signal value of the gene chip is positive. Each typing probe selects non-influenza virus (i.e., negative strain) and blank control for gene chip hybridization, and through repeated experiments and data statistics, the background statistical average value +2SD of negative strain and blank control is used as each The cutoff value of the probe is shown in Table 6. The discrimination ability of each mutation detection probe is more than 2.5 times as the criterion for judging whether there is a mutation at the drug resistance site.

[0061] Table 6 Determination of the Cutoff value of each probe

[0062]

Embodiment 3

[0063] Example 3: Influenza A virus nucleic acid screening and gene chip specificity evaluation for drug resistance detection

[0064] Specificity is the most important assessment index of a diagnostic method. Using the optimized system and conditions, the gene chip of the present invention has detected 24 strains of various types of influenza viruses, parainfluenza viruses, and common respiratory viruses. The test results are shown in the attached image 3 . As can be seen from the figure, the present invention can not only correctly distinguish influenza A virus from other common respiratory tract viruses and influenza B virus, but also distinguish influenza A H1N1, seasonal H1N1, and seasonal H3N2 among influenza A viruses. It can be correctly distinguished from avian influenza H5N1 with good specificity. In addition, a seasonal H1N1 influenza virus Tamiflu resistance mutation site was found to be mutated in the experiment. After sequencing verification, the result was con...

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Abstract

The invention relates to a gene chip for determination and drug resistance detection of influenza A virus. The preparation method of the gene chip comprises the steps of preparing a universal primer, preparing a subtype nucleic acid parting probe and a drug resistance probe, preparing an oligonucleotide chip, establishing an RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) system and establishing a hybrid system. The gene chip prepared by the method disclosed by the invention can be used for simultaneously determining five kinds of subtype influenza A viruses, including influenza A H1N1 influenza virus, seasonal H1N1 influenza virus, seasonal H3N2 influenza virus, poultry H5N1 influenza virus and swine H1N1 influenza virus. In addition, the gene chip can be used for prompting the condition that the influenza A virus is non-drug resistant, has the advantages of quickness, accuracy, high throughput and high specificity and can be used for providing guidance for monitoring, clinical diagnosis and treatment of the influenza viruses.

Description

technical field [0001] The invention relates to the preparation and application of a gene chip for influenza A virus screening and drug resistance detection, and belongs to the technical field of gene chip detection. Background technique [0002] Influenza virus belongs to Orthomyxoviridae in virus taxonomy. Its genome is segmented single-stranded negative-sense RNA. Influenza viruses are divided into three types, A, B, and C, according to the antigenic characteristics of the nucleoprotein (NP) and membrane protein (MP) and their genetic characteristics. Influenza A viruses can be divided into many subtypes according to their surface hemagglutinin (HA) and neuraminidase (NA) protein structures and their genetic characteristics. So far, influenza A viruses have discovered 16 subtypes of hemagglutinin (H1-16), neuraminidase has 9 subtypes (N1-9). Since the genome is segmented, gene reassortment between different strains of the same type is prone to occur. In particular, th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 王升启刘琪琦陈苏红张敏丽刘志红朱坤
Owner 深圳市普瑞康生物技术有限公司
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