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Nuclei acid detection kit for RNA isothermal amplification avian influenza virus H7N9 (2013)

A kind of avian influenza virus, H7N9 technology, applied in the field of in vitro diagnostic reagents

Active Publication Date: 2015-03-25
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] TMA and SAT technologies can directly amplify RNA, which can be detected quickly, with high sensitivity and high specificity. There is no report on the H7N9 kit of TMA.

Method used

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  • Nuclei acid detection kit for RNA isothermal amplification avian influenza virus H7N9 (2013)
  • Nuclei acid detection kit for RNA isothermal amplification avian influenza virus H7N9 (2013)
  • Nuclei acid detection kit for RNA isothermal amplification avian influenza virus H7N9 (2013)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Development and optimization of detection reagents for avian influenza virus H7N9 (2013) nucleic acid detection kit

[0029] 1. Design of capture probes (TCO), primers and target nucleic acid detection probes:

[0030] Through the sequence comparison analysis of the existing avian influenza virus H7N9 (2013) nucleic acid sequences in the Genbank database, the HA conserved gene fragment and the NA conserved gene fragment of the avian influenza virus H7N9 (2013) were used as amplification target sites. Design primers and probes for highly conserved segments with no secondary structure.

[0031] 2. Establishment and optimization of extraction system and reaction system:

[0032] The extraction system and reaction system of the present invention refer to patents ZL 200810111479.0 and ZL 200810111478.6, except for capture probe (TCO), primer and detection probe sequence, concentration and reaction time. Therefore, the present invention mainly screens capture pro...

Embodiment 2

[0043] Example 2 Composition and detection of avian influenza virus H7N9 (2013) nucleic acid detection kit

[0044] 1. Prepare a kit comprising the following components:

[0045] The kits are divided into the specimen processing unit A box and the nucleic acid amplification detection unit B box. Box A Including virus preservation solution, nucleic acid extraction solution, washing solution; B box Including H7N9 (2013) reaction solution, H7 detection solution, N9 detection solution, SAT enzyme solution, H7N9 (2013) positive control, H7N9 (2013) negative.

[0046] specific:

[0047] Virus preservation solution: a solution containing 50mM Tris (pH7.0), 0.1% (V / V) SDS, 1% (V / V) Triton X-100, 0.1% (V / V) NP-40.

[0048] Nucleic acid extraction solution: a solution containing 250mg / mL magnetic beads and 0.25μM capture probe (TCO). The capture probe sequence is: 5'accaaccaacaatttgagttgatagacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa'3.

[0049] Washing solution: a solution containing 150 ...

Embodiment 3

[0100] Example 3 Sensitivity Detection of Avian Influenza Virus H7N9 (2013) Nucleic Acid Detection Kit

[0101] The kit described in Example 2 was used to detect RNA transcribed in vitro from H7 and N9. The main raw material SAT enzyme solution used in the examples and the in vitro transcribed RNA of the positive control were provided by RD Biosciences in the United States. The 7500 PCR instrument was a product of ABI in the United States. Reagents such as NTPs and dNTPs and other instruments were commercially available products.

[0102] Set the concentration to 1 x 10 6 copies / μl of HA gene in vitro transcribed RNA and 1×10 6 Copies / μl NA gene was transcribed RNA in vitro, diluted in a 10-fold gradient respectively, and detected according to the steps described in Example 2.

[0103] from figure 1 with figure 2 The test results show that the detection sensitivity of both H7 and N9 can reach 10copies / reaction, and the sensitivity of the whole kit is 10copies / reaction, in...

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Abstract

The invention relates to a nucleic acid detection kit for RNA isothermal amplification avian influenza virus H7N9 (2013). The kit includes a virus preservation solution, a nucleic acid extraction solution, a washing liquid, an H7N9 reaction liquid (2013), an H7 detection liquid, an N9 detection liquid, an SAT enzyme liquid, an H7N9 (2013) positive control reagent, and an H7N9 (2013) negative control reagent. The detection kit provided by the invention has the characteristics of high specificity, high sensitivity, low pollution and rapid detection, and plays an important role in clinical diagnosis of early infection of avian influenza virus H7N9 (2013).

Description

technical field [0001] The invention relates to the technical field of in vitro diagnostic reagents, in particular to a kit for detecting avian influenza virus H7N9 (2013) by using magnetic bead-RNA enrichment technology to extract and purify target RNA and constant temperature nucleic acid simultaneous amplification detection technology (SAT). The detection kit of the invention can realize the detection of avian influenza virus H7N9 (2013) in secretions such as poultry feces and secretions such as human throat and nose. Background technique [0002] The avian influenza virus belongs to the Orthomyxoviridae Influenza A virus genus, and the virus particles are pleomorphic, in which the spherical diameter is 80-120nm and has a capsule. In addition to infecting poultry, avian influenza A virus can also infect humans, pigs, horses, mink and marine mammals. The H7N9 human infection with avian influenza in 2013 is a new type of reassortant virus, and its internal gene comes from ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6844C12Q2521/107C12Q2521/119C12Q2563/107
Inventor 尹华立白立志马道亮于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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