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Kit for detecting drug-resistant site of influenza viruses A (IFVA), preparation thereof and application thereof

A technology of influenza A virus and detection kits, applied in the determination/testing of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of long operation cycle, inconvenient hospital development and promotion, expensive detection equipment, etc.

Inactive Publication Date: 2012-03-14
上海之江生物医药科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the gold standard for detecting gene mutations is gene sequencing. This method requires expensive detection equipment, and the operation cycle is long. It usually takes 2-3 days to produce results, which is not convenient for most hospitals in China to carry out and promote.

Method used

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  • Kit for detecting drug-resistant site of influenza viruses A (IFVA), preparation thereof and application thereof
  • Kit for detecting drug-resistant site of influenza viruses A (IFVA), preparation thereof and application thereof
  • Kit for detecting drug-resistant site of influenza viruses A (IFVA), preparation thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The preparation of embodiment 1 kit

[0050] 1) Preparation of IFVA-H274Y nucleic acid fluorescent PCR detection mixture:

[0051] Mix IFVA upstream primer 0.75 μl / test; downstream primer 0.75 μl / test; probe 0.05 μl / test; RT-PCR MIX 12.5 μl / test; liquid.

[0052] Upstream primer: 5'-tggacaggcctcatacaaga-3' (SEQ ID NO: 2)

[0053] Downstream primer: 5'-attcgagccatgccagttat-3' (SEQ ID NO: 3)

[0054] Probe: 5'-FAM-CCtCAtaGtAAtaa-BHQ1-3' (SEQ ID NO: 4)

[0055] The above primers and probes were synthesized by conventional methods.

[0056] 2) The IFVA-H274Y fluorescent PCR detection mixture, RT-PCR enzyme, positive control substance and negative control substance are individually packaged and assembled into a kit.

[0057] Positive control substance: H274Y mutant strain constructed by genetic engineering

[0058] The preparation method of positive control substance is:

[0059] Use the above upstream and downstream primers (SEQ ID NO: 2 and SEQ ID NO: 3) to amplify ...

Embodiment 2

[0065] Application of embodiment 2 detection kit

[0066] Adopt the kit prepared by embodiment 1

[0067] Specimen 1, 2, and 3 were obtained from clinical specimens.

[0068] 2.1 Processing of test specimens:

[0069] Take 100 μl of the sample and place it in a 1.5ml centrifuge tube, add 300 μl of lysate (Trizol), and then add 100 μl of chloroform, shake and mix on a mixer for 5 sec or invert for 15 times. Centrifuge at 13,000 rpm for 15 min. Aspirate the upper layer liquid, add an equal volume of isopropanol, invert and mix well, and let stand at room temperature for 10 minutes. Centrifuge at 13,000 rpm for 10 min, pour off the supernatant; add 700 μl of 75% ethanol, and wash upside down. Centrifuge at 13,000rpm for 5min, place it upside down on absorbent paper, and discard the liquid. Centrifuge at 4,000rpm for 5sec, shake the residual liquid on the tube wall to the bottom of the tube, use a micro-sampler to absorb the liquid as much as possible, add 30μl DEPC-H 2 O di...

Embodiment 3

[0084] The specificity research of embodiment 3 detection kits

[0085] Adopt the kit prepared by embodiment 1

[0086] Influenza A virus H1 subtype specimens, influenza A virus H3 subtype specimens, influenza B virus specimens, enterovirus 71 specimens obtained clinically; negative cell culture supernatants of influenza A H1N1 influenza virus culture cell line MDCK 1 serving. The sample numbers are N1, N2, N3, N4 and N5, respectively.

[0087] 3.1 Processing of test specimens:

[0088] Take 100 μl of the sample and place it in a 1.5ml centrifuge tube, add 300 μl of lysate (Trizol), and then add 100 μl of chloroform, shake and mix on a mixer for 5 sec or invert for 15 times. Centrifuge at 13,000 rpm for 15 min. Aspirate the upper layer liquid, add an equal volume of isopropanol, invert and mix well, and let stand at room temperature for 10 minutes. Centrifuge at 13,000 rpm for 10 min, pour off the supernatant; add 700 μl of 75% ethanol, and wash upside down. Centrifuge a...

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Abstract

The invention discloses a kit for detecting the drug-resistant site of IFVA, a preparation thereof and an application thereof. The kit for detecting the drug-resistant site of the IFVA comprises a mixed IFVA-H274Y fluorescent PCR detection solution, an RT-PCR enzyme, a positive comparison product, and a negative comparison product, wherein the mixed IFVA-H274Y fluorescent PCR detection solution comprises primers, a probe, RT-PCR MIX and water. The kit for detecting the drug-resistant site of the IFVA is used for the drug-resistant analysis and discrimination of the IFVA to Tamiflu.

Description

technical field [0001] The invention relates to an influenza A virus drug-resistant site detection kit and a preparation method and use thereof. Background technique [0002] Influenza A virus is a common influenza virus. According to the difference of H and N antigens, influenza A virus can be divided into many subtypes. H can be divided into 15 subtypes (H1~H15), and N has 9 subtypes (N1~H15). N9). Influenza A virus is most likely to mutate, and an influenza pandemic is caused by the emergence of a new subtype or the reappearance of an old subtype of influenza A virus. Influenza is characterized by recurrent, unpredictable local epidemics and rare global pandemics. Since 1889, there have been several worldwide pandemics caused by antigenic mutations of influenza A virus. Influenza epidemics are accompanied by an increase in mortality. In the influenza epidemic study in the United States from 1972 to 1995, influenza epidemics occurred in 19 out of 23 years and caused ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 朱旭平邵俊斌刘燕朱勤玮
Owner 上海之江生物医药科技有限公司
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