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H3/H1N1(2009)/H1N1 influenza A virus multiplex fluorescence PCR detection kit and application thereof

A technology for detection kits and influenza viruses, which is applied in the field of multiple fluorescent PCR detection kits and virus detection kits. It can solve the problems that infections cannot be effectively distinguished, and consumes a lot of time and energy, and achieves short detection time and high accuracy. high effect

Inactive Publication Date: 2017-12-08
BEIJING MOKOBIO LIFE SCI CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional fluorescent real-time PCR can only amplify the nucleic acid of one pathogen at a time. It takes multiple attempts to accurately identify the causative pathogen, which consumes a lot of time and energy, and it cannot effectively distinguish infections caused by multiple pathogens

Method used

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  • H3/H1N1(2009)/H1N1 influenza A virus multiplex fluorescence PCR detection kit and application thereof
  • H3/H1N1(2009)/H1N1 influenza A virus multiplex fluorescence PCR detection kit and application thereof
  • H3/H1N1(2009)/H1N1 influenza A virus multiplex fluorescence PCR detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Assembling of a multiplex fluorescence PCR detection kit for influenza A H3 / H1N1(2009) / H1N1 influenza virus nucleic acid

[0035] 1) Design primer / probe combination:

[0036] According to the nucleic acid sequence of each pathogen, sequence comparisons between the same species were performed to find conserved regions, and Primer Express 3.0 software was used to design primers and Taqman probes for multiple PCR on these conserved regions. Perform BLAST analysis on the NCBI website for each primer / probe combination obtained to ensure that it does not cross-react with other microorganisms that may be present in the sample. Finally, its performance is verified through experiments. The design of the internal control uses the same method. The designed primers / probes are shown in Table 1 below. The synthesis was completed by Shenggong Bioengineering (Shanghai) Co., Ltd.

[0037] Table 1 Primers and probes used to amplify A H3, new A H1N1 (2009), seasonal H1N1 influenza ...

Embodiment 2

[0050] Example 2 Detection of influenza A H3, new A H1N1 (2009) and seasonal H1N1 influenza viruses respectively

[0051] The isolated and cultured strains of influenza A H3, new A H1N1 (2009) and seasonal H1N1 influenza viruses were used respectively, and the samples were tested with the kit prepared in Example 1.

[0052] The QIAamp MinElute Virus Spin Kit (QIAGEN) was used to extract nucleic acids from the samples. Then add H3 / H1N1(2009) / H1N1 primer probe mixture (2μL), enzyme system (2μL) and nucleic acid extracted from the sample or negative / positive control reagent (5μL) into 16μL of PCR reaction buffer. Subsequently, each reaction tube was subjected to PCR reaction on a suitable real-time fluorescent PCR machine (such as ABI 7500, Applied Biosystems) according to the following procedures: 50℃ for 25min, 95℃ for 5min; 95℃ for 10s, 55℃ for 15s, 72℃ Maintained for 30s, 5 cycles; 95°C for 10s, 60°C for 45s (collecting fluorescence signal), 40 cycles.

[0053] Result judgment:

[...

Embodiment 3

[0073] Example 3 One-time detection of influenza A H3, new A H1N1 (2009) and seasonal H1N1 influenza viruses

[0074] 1. Extraction of viral nucleic acid

[0075] The QIAamp MinElute Virus Spin Kit (QIAGEN) was used to extract viral nucleic acids from influenza A H3, new A H1N1 (2009) and seasonal H1N1 influenza virus samples. Perform a mixing operation on the extracted viral nucleic acid samples at the same ratio. Five gradients of 10-fold dilution are used as templates for detection.

[0076] 2. PCR amplification detection:

[0077] 1) Prepare PCR reaction solution according to the following composition (n is the number of reaction tubes).

[0078] The PCR reaction buffer is 16 μL×n, the H3 / H1N1(2009) / H1N1 primer probe mixture is 2 μL×n, and the enzyme system is 2 μL×n. (Note: Before use, ensure that the PCR reaction buffer, H3 / H1N1(2009) / H1N1 primer probe mixture is fully dissolved and mixed, and the enzyme system needs to be centrifuged before use to ensure that all enzymes are c...

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Abstract

The invention provides a one-tube multiplex fluorescence PCR detection method for A H3, novel A H1N1 (2009) and seasonable H1N1 influenza viruses. The method adopts a primer with the sequence as shown in SEQ ID No:1-9 and a probe. The invention further provides an H3 / H1N1(2009) / H1N1 influenza A virus multiplex fluorescence PCR detection kit. The kit comprises the primer and the probe. The H3 / H1N1(2009) / H1N1 specific primer and the Taqman probe are adopted, and FAM / HEX / ROX multiplex fluorescein labeling is used to realize multiplex detection of the A H3, novel A H1N1 (2009) and seasonable H1N1 influenza viruses. The kit has the advantages of high specificity and sensitivity, high speed, simple and convenient operation, low cost and the like are achieved, and can serve as a multiplex detection reagent for scientific research and clinical application.

Description

Technical field [0001] The invention relates to a virus detection kit, in particular to a multiple fluorescent PCR detection kit capable of simultaneously detecting influenza A H3, new A H1N1 (2009) and seasonal H1N1 influenza viruses, belonging to the technical field of nucleic acid detection. Background technique [0002] Influenza virus (influenza virus, abbreviated as influenza virus) is the pathogen of influenza (influenza, abbreviated as influenza). Influenza viruses belong to the Orthomyxoviridae family, and their genome is a segmented single-stranded negative-strand RNA. Influenza virus hemagglutinin is located on the influenza virus envelope and mediates the adsorption and penetration of influenza virus into host cells. According to the characteristics of M protein and NP protein, influenza viruses are divided into types A (A), B (B), and C (C). In addition to infecting humans, influenza A viruses can also infect animals such as poultry, pigs, and horses. And often cau...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2537/143
Inventor 范建张秀芬徐宁金鑫
Owner BEIJING MOKOBIO LIFE SCI CO LTD
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