Gene chip for detection and typing of bird flu virus

A bird flu virus, gene chip technology, applied in the field of biochip

Inactive Publication Date: 2007-08-22
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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  • Gene chip for detection and typing of bird flu virus
  • Gene chip for detection and typing of bird flu virus
  • Gene chip for detection and typing of bird flu virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Design and synthesis of embodiment 1 primer

[0044] 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 Designer2.1 software. Shanghai Sangon Bioengineering Co., Ltd. Synthesized by the company, the primer sequences are shown in Table 1.

[0045] Table 1. Primers for multiplex PCR / RT-PCR

[0046] type

[0047] N2-R

[0048] Note: Y=(C, T), W=(A, T). Note: Y=(C, T), W=(A, T).

Embodiment 2

[0049] Embodiment 2 can obtain the RT-PCR amplification of the avian influenza subtype of standard strain

[0050] 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.

[0051] 2.1 Extraction of template RNA

[0052] 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.

[0053] 2.2 RT-PCR reaction system

[0054] 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 ...

Embodiment 3

[0056] Embodiment 3 can not obtain the artificial synthesis of the corresponding gene fragment of the avian influenza virus genotype subtype of standard strain

[0057] 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.

[0058] 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.

[0059] 3.1 Primer design

[0060] 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 present invention relates to gene chip for the detection and subtyping of bird flu virus. The present invention establishes gene chip with high specificity, high sensitivity and great practicability for detection and subtyping of bird flu virus through designing and screening 50 pairs of specific primers, 2 universal primers, 52 specific probes and 3 quality control probes for the multiple RT-PCR subtyping detection of bird flu virus genes based on the gene sequences of different bird flu virus subtypes Genebank reports; and performing the effectiveness test, specificity test, sensitivity test and condition optimization of gene chip detection. The present invention establishes also the detection and subtyping process with the chip and gives the initial 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 Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 韩雪清林祥梅陈洪俊刘建梅琳黄金海侯义宏
Owner 中国检验检疫科学研究院动植物检疫研究所
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