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Fluorescent quantitative PCR primer, probe, kit and detection method for detecting avian influenza subtype

An influenza virus, multiple fluorescence quantitative technology, applied in the field of bioengineering, can solve problems such as low sensitivity, achieve high sensitivity, shorten detection time and detection times, and avoid cumbersome operations.

Active Publication Date: 2017-02-22
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Qin Zhifeng et al. conducted a simultaneous integrated rapid detection of highly pathogenic H5, H7, and H9 subtypes of avian influenza viruses. Sex is 1000, 1000, 500 copy numbers respectively, the sensitivity is low

Method used

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  • Fluorescent quantitative PCR primer, probe, kit and detection method for detecting avian influenza subtype
  • Fluorescent quantitative PCR primer, probe, kit and detection method for detecting avian influenza subtype
  • Fluorescent quantitative PCR primer, probe, kit and detection method for detecting avian influenza subtype

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preparation example Construction

[0068] In the present invention, the preparation method of the positive plasmid standard substance preferably comprises the following steps:

[0069] A, extract the total RNA of H5, H7 and H9 subtype influenza virus samples respectively;

[0070] B. The total RNA of the H5, H7 and H9 subtype influenza viruses obtained in the step A is respectively reverse-transcribed to obtain the cDNA of the H5, H7 and H9 subtype influenza viruses;

[0071] C, using the cDNA obtained in step B as a template, using the nucleotide sequences shown in SEQ ID No.13 and SEQ ID No.14 as upstream primers and downstream primers to amplify H5 subtype influenza virus-specific target fragments , using the nucleotide sequences shown in SEQ ID No.15 and SEQ ID No.16 as upstream primers and downstream primers to amplify H7 subtype influenza virus-specific target fragments, using SEQ ID No.17 and SEQ ID No.18 The nucleotide sequences shown are respectively used as upstream primers and downstream primers to ...

Embodiment 1

[0109] Primer design

[0110] According to the M gene of AIV in GenBank and the hemagglutinin gene sequences of H5, H7 and H9 subtype avian influenza viruses, DNAMAN software was used for homology analysis, and Primer Express5.0 software was used to design specific primers and TaqMan in its conserved regions. fluorescent probe. Wherein, M gene primers and probes are obtained according to laboratory operation standards of avian influenza virus. The designed primer pairs and probes of H5, H7 and H9 subtype avian influenza viruses and M gene primer pairs and probes were entrusted to Shanghai Biological Engineering Company to synthesize.

[0111] RNA extraction

[0112] Take an appropriate amount of chicken embryo allantoic fluid, centrifuge at 12,000 rpm for 5 minutes, take 500 μl of supernatant, add 700 μl of Trizol to it, shake and mix, let stand for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes, take the supernatant and add it to 500 μl of isopropanol, mix w...

Embodiment 2

[0120] Dilute the recombinant plasmid of the universal vector according to a 10-fold gradient to obtain 1×10 0 Copy number / μl~1×10 7 Copy number / μl serially diluted plasmids were detected and analyzed in order to obtain the lower limit of detection. Obtain the Ct values ​​of different concentrations of standard products to determine the minimum copy number of templates that can be detected by this method. At the same time, use the above-mentioned different concentrations of templates for detection by conventional PCR methods, and identify PCR products by 1% agarose gel electrophoresis. The amplification curve of each specific target fragment is as follows Figure 5-8 shown.

[0121] The study found that when the Ct value > 35 cycle is negative, and the Ct value ≤ 35 cycle is positive. The detection limit of the established fluorescent quantitative PCR method for detecting H5, H7, H9 influenza virus and influenza virus M gene was 10 copies.

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Abstract

The invention provides a multiplex fluorescent quantitative PCR primer and a probe for detecting H5, H7 and H9 subtype avian influenza viruses. With adoption of the primer and the probe, multiplex fluorescent quantitative PCR can be adopted to simultaneously detect which subtypes the H5, H7 and H9 subtype avian influenza viruses concretely are, no influences exist among the primers of different subtypes, the specificity is strong, the detection sensitivity is 10-50 copy numbers, a target sequence can be accurately detected quantitatively and qualitatively, the repeatability is good, and the reliability is high. The invention provides a multiplex fluorescent quantitative PCR kit for simultaneously detecting the H5, H7 and H9 subtype avian influenza viruses. The kit has the detection sensitivity being 10-50 copy numbers and is high in sensitivity and strong in specificity. The invention also provides a detection method which is good in stability. With adoption of plasmids with gene fusion as a positive standard substance, tedious operation of changing the positive standard substance for multiple times is avoided, the detection time is greatly shortened, the detection times are greatly reduced, and detection of a sample can be finished within 2h.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a fluorescent quantitative PCR primer, a probe, a kit and a detection method for detecting avian influenza virus. Background technique [0002] Influenza virus (influenza virus) belongs to Orthomyxoviridae, and its genome is segmented single-stranded negative-sense RNA. According to the characteristics of M protein and NP protein, influenza virus is divided into A (A), B (B), and C ( Type C), wherein the type A influenza virus can infect poultry in addition to infecting humans, so that the infected poultry suffers from avian influenza (AI). According to the difference of hemagglutinin (HA) and neuraminidase (NA) antigens of AIV, AIV can be divided into 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9). The 1918 Spanish flu was caused by the H1N1 subtype and caused about 50 million deaths; the 1957 "Asian flu" caused by the H2N2 subtype caused about 2.8 million...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2537/143C12Q2545/114C12Q2563/107C12Q2531/113
Inventor 周继勇闫丽萍刘俊丽雷静粟硕胡伯里
Owner NANJING AGRICULTURAL UNIVERSITY
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