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Double droplet type digital PCR absolute quantification detection kit for AIV H7N9 subtypes

A detection kit, H7N9 technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of easy mutation of bird flu virus, lagging of AIVH7N9 detection work, etc., to achieve high accuracy, Easy to operate and highly specific effects

Inactive Publication Date: 2016-02-17
BEIJING SEAGULL BIOVENTURES & BIOTECH CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the application of ddPCR in the absolute quantitative detection of avian influenza virus. Although ddPCR is expected to be used in cancer screening, diagnosis of infectious diseases, quantification of genetically modified ingredients in food, and water quality monitoring, due to the current tendency of avian influenza virus to mutate In particular, the H7N9 subtype of human-infected avian influenza virus that occurred in my country in 2013 was a new mutant gene, which caused a lag in the detection of the newly discovered AIV H7N9, and it was impossible to effectively achieve accurate, accurate, sensitive, and accurate detection of this subtype of virus. Efficient and specific detection

Method used

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  • Double droplet type digital PCR absolute quantification detection kit for AIV H7N9 subtypes
  • Double droplet type digital PCR absolute quantification detection kit for AIV H7N9 subtypes
  • Double droplet type digital PCR absolute quantification detection kit for AIV H7N9 subtypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 detects the design of the specific primer and probe of avian influenza H7N9 subtype

[0041] 1. Design of primers and probes of the present invention: the hemagglutinin gene (HA gene, GenBank accession number is KP417119) of avian influenza H7N9 (AIV-H7N9) as the target gene, and the nerve gene of avian influenza H7N9 (AIV-H7N9) The amino acid enzyme gene (NA gene, GenBank accession number KR351266) was used as the target gene, and specific primers suitable for ddPCR were designed.

[0042] This example gives the process of screening the best primers, select several pairs of alternative primers designed by software for screening, the alternative primers are as follows, see Table 1.

[0043] Table 1

[0044]

[0045]

[0046] 2. Screening of primers

[0047] (1) The HA primers were randomly matched into four pairs: F1R1, F1R2, F2R1, F2R2; the NA primers were randomly matched into four pairs: F1R1, F1R2, F2R1, F2R2.

[0048] (2) Then reverse transcri...

Embodiment 2

[0055] Example 2 Optimization of the annealing temperature of the PCR method for detecting H7N9 on the ddPCR platform.

[0056] 1. Select the combination of primers and probes as: HA-F2R2P2+NA-F2R2P2.

[0057] 2. Then on the ddPCR platform, ddPCR system formula: 2XOne-stepRT-ddPCRSupermix 10μl, upstream primer, downstream primer 1μl, probe 0.5μl, RNaseFreedH 2 O3μl, positive template 2μl, total volume 20μl (concentration of both primers and probes is 10μM). Do 8 replicate wells and generate microdroplets.

[0058] 3. Amplify on a PCR instrument. The PCR amplification program is reverse transcription at 50°C for 10 minutes; pre-denaturation at 95°C for 10 minutes; denaturation at 94°C for 30 sec, and annealing at 50-60°C (8 temperature gradients are automatically distributed by the instrument) for 60 sec, a total of 40 sec. cycle; 98 ° C for 10 min to end the reaction. Detection on the droplet digital PCR detector.

[0059] 4. Analyzing results: Select an annealing temperat...

Embodiment 3

[0060] The optimization experiment of embodiment 3 primers, probe concentration

[0061] The concentrations of the designed primers and probes were all 10 μM, and the combination was HA-F2R2P2+NA-F2R2P2. The system configuration method is shown in Table 2.

[0062] Table 2

[0063]

[0064]

[0065] Then micro-droplets were generated on the ddPCR platform, transferred to a 96-well plate, and sealed with an aluminum film.

[0066] Amplify on a PCR instrument. The amplification program of PCR is reverse transcription at 50°C for 10 minutes; pre-denaturation at 95°C for 10 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 60 seconds, a total of 40 cycles; and 98°C for 10 minutes to end the reaction. Detection on the droplet digital PCR detector.

[0067] Analysis results: According to the experimental results, the formula of HA-F2R2P2+NA-F2R2P2 primers and probes was selected, and the selected primers were 1.8 μl and the probes were 0.4 μl respectively. ...

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Abstract

The invention provides a double droplet type digital PCR absolute quantification kit for AIV H7N9 subtypes. The double droplet type digital PCR absolute quantification kit comprises two pairs of specific primer pairs and two specific probes used in cooperation with specific primers, and nucleotide sequences are as shown in SEQ ID No.1-6 respectively. According to the double droplet type digital PCR absolute quantification kit, AIV H7N9 subtypes can be detected quickly, specifically and sensitively through a digital PCR instrument, and the detection sensitivity reaches one copy. The double droplet type digital PCR absolute quantification kit is simple in use method, low in cost, capable of detecting the AIV H7N9 subtypes quickly, directly and quantitively without a standard curve, very applicable to disease supervision, on-site emergency detection and clinical sample detection, and suitable for application and popularization in a wide range.

Description

technical field [0001] The invention relates to the technical field of kit detection, in particular to a double droplet type digital PCR absolute quantitative detection kit for detecting subtypes of avian influenza virus H7N9. Background technique [0002] Avian influenza virus subtype H7N9 (AIVH7N9) is an influenza A virus, a subtype of avian influenza virus. Since the human cases of H7N9 avian influenza infection came out in 2013, the China Hangzhou Center for Disease Control and Prevention and the WHO China Influenza Center have collected and analyzed the genomes of four new H7N9 avian influenza A, and published them on the GISAID database. Using these 4 genomes and 1193 known genomes of each subtype of influenza, a comprehensive phylogenetic tree and evolutionary history were analyzed. The results showed that among the eight genes of the new H7N9 subtype avian influenza, the surface hemagglutinin (Haemagglutinin, H) protein gene came from the H7 subtype virus, and the n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2545/113C12Q2537/143C12Q2563/159
Inventor 陈晨冯小宇韦海涛宋彦军刘晓东周德刚马付坤闫慧张静依
Owner BEIJING SEAGULL BIOVENTURES & BIOTECH CO LTD
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