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Primer system and method for detecting and analyzing avian influenza virus

A technology of avian influenza virus and universal primers, applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc.

Inactive Publication Date: 2012-05-23
中国检验检疫科学研究院动植物检疫研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Conventional PCR / RT-PCR technology can only detect and diagnose one pathogen in one PCR reaction. , it is necessary to carry out multiple PCR / RT-PCR reactions to finally confirm the diagnosis

Method used

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  • Primer system and method for detecting and analyzing avian influenza virus
  • Primer system and method for detecting and analyzing avian influenza virus
  • Primer system and method for detecting and analyzing avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Design and synthesis of embodiment 1 primer

[0040] According to the genome sequences of different subtypes of avian influenza viruses published in Genebank, DNAStar / Bioedit software was used for sequence analysis, and 25 pairs of primers were designed using Primer5.0, OMIGA and Beacon Designer 2.1 software. Shanghai Sangon Bioengineering Co., Ltd. Synthesized, the sequence is shown in Table 1.

[0041] type Primer number Sequence (5'~3') The length of the amplified fragment H1 H1-F H1-R GGAGCAATTGAGTTCAGTATCGACACTCTCCTATTGTGACTG 601bp H3 H3-F H3-R TGTTACCCTTATGATGTGCCCCCTGTTGCCAATTTCAGAG 669bp H5 H5-F H5-R AGTGAATTGGAATATGGTAACTG AACTGAGTGTTCATTTTGTCAAT 380bp H6 H6-F H6-R AAGGCACTTATTGGRTCAGGGTCCTCTAGTTTCAATCTGTGG 685bp H7 H7-F H7-R TCAGGWTCTTCWTTCTATGCTCYCCTTGTGCATTTTGATG 641bp H9 H9-F H9-R AAGAGAATGGTCCTACATCGT GGATCTTACTCGCAATGTCTG 493bp N1 N1-F N1-R TCCCACTTGGAATGCAGAA...

Embodiment 2

[0044] Embodiment 2 can obtain the RT-PCR amplification of the avian influenza subtype of standard strain

[0045]In this example, for the 8 avian influenza virus subtypes (H1, H3, H5, H6, H7, H9, N1 and N2) that can obtain standard strains, RNA is extracted from the standard strains and then used as a template Directly proceed to conventional RT-PCR amplification.

[0046] 2.1 Extraction of template RNA

[0047] According to the method provided by the instructions of QIAamp Viral RNA Mini Kit (purchased from QIAGEN Company), the genomic RNA of avian influenza virus was extracted from the standard strain of avian influenza virus.

[0048] 2.2 RT-PCR reaction system

[0049] Refer to the operating instructions of the one-step RT-PCR kit (purchased from QIAGEN) with slight modifications. Reaction system: Add QIAGENE onestep RT-PCR buffer (5×) 10μL, 10mM dNTP 2.0μL, Enzyme mix 2.0μL, RNase inhibitor 0.5μL, upstream and downstream primers 0.5μL (10μM), RNA Template 5 μL, add R...

Embodiment 3

[0051] Embodiment 3 can not obtain the artificial synthesis of the corresponding gene fragment of the avian influenza virus genotype subtype of standard strain

[0052] In this embodiment, for the remaining 17 subtypes (including H2, H4, H8, H10, H11, H12, H13, H14, H15, H16, N3, N4, N5, N6, N7, N8) that cannot obtain standard strains and N9), the corresponding target DNA fragments were synthesized by bridging PCR amplification.

[0053] In this example, the target DNA fragments of all the above-mentioned 17 genotypes were synthesized. Here, only H15 is used as an example to describe the synthesis process in detail. For the synthesis process of the target DNA fragments of the remaining 16 genotypes, in addition to selecting corresponding different primers, All other steps are the same.

[0054] 3.1 Primer design

[0055] Using DNAStar and Bioedit software to analyze the full gene sequence of all H15 type AIVs that have been published, and using Primer5.0 and OMIGA software t...

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Abstract

The invention provides a method for detecting fowl influenza virus and parting primer system and augmenting fowl influenza virus RNA asymmetrically. The invention designs and screens 25 pairs multiple asymmetrical RT-PCR primers, a pair general primer, 52 specific probes and 3 quality control probes according to the report fowl influenza virus total hypotype genome sequence in Genebank, wherein every pair in 25 pairs multiple asymmetrical RT-PCR primers is fit for a hypotype of fowl influenza virus, the primer system comprising 25 pairs multiple asymmetrical RT-PCR primers and a pair general primer can be used for detecting and parting fowl influenza virus, the primer system builds the method of asymmetrically augmenting fowl influenza virus RNA and detecting and parting fowl influenza virus. The invention provides strong specificity, high sensibility, which also proceeds the first step application.

Description

Technical field: [0001] The invention belongs to the field of biological chips. Specifically, the present invention relates to a gene chip for detecting and typing all subtypes of avian influenza virus, preparing type-specific probes and quality control probes used in the gene chip, and using the gene chip to detect and typing methods, as well as multiple RT-PCR primers, multiple asymmetric RT-PCR primers and universal primers used in detection and typing research. Background technique [0002] Avian Influenza (AI) is a general term for poultry infections and diseases caused by influenza A viruses of the genus Influenzavirus. Poultry such as chickens, turkeys, ducks, and quails, as well as wild birds, waterfowl, and seabirds can be infected, and the harm caused to domestic chickens and turkeys is the most serious. The main reason for the worldwide epidemic of influenza, pigs can also be used as "mixers" to spread the disease. Avian influenza virus (Avian Influenza virus,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12N15/10
Inventor 韩雪清林祥梅吴绍强梅琳朱忠武廉慧峰贾广乐
Owner 中国检验检疫科学研究院动植物检疫研究所
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