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Avian influenza virus H5, H7 and H9 subtype triple fluorescent quantitative RT-PCR detection kit and application thereof

The technology of a bird flu virus and detection kit is applied in the determination/testing of microorganisms, recombinant DNA technology, DNA/RNA fragments, etc., which can solve the problems of achieving sensitivity, achieve high sensitivity, good application potential and application value, low cost effect

Pending Publication Date: 2021-01-05
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, comprehensive optimization is obviously difficult to make a specific single reaction reach the sensitivity of its single-plex fluorescent quantitative PCR reaction.

Method used

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  • Avian influenza virus H5, H7 and H9 subtype triple fluorescent quantitative RT-PCR detection kit and application thereof
  • Avian influenza virus H5, H7 and H9 subtype triple fluorescent quantitative RT-PCR detection kit and application thereof
  • Avian influenza virus H5, H7 and H9 subtype triple fluorescent quantitative RT-PCR detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Design of Primers and Probes for Avian Influenza H5, H7, H9 Subtypes

[0062] This example refers to the hemagglutinin-related gene sequences of the H5, H7 and H9 subtypes of avian influenza virus published in the GenBank database, and uses the DNAMAN software to conduct a comparative analysis of gene homology to determine its nucleic acid conserved sequence region as a specific target fragment. Using Primer Express5.0 software, design specific primers and fluorescent probes for their specific target fragments. Experimental comparison proves that the preferred specific primers and fluorescent probes have a significant positive effect on improving the specificity of the reaction.

[0063] In the present invention, the specific probe of H5 subtype avian influenza virus HA gene carries Cy5 fluorescent reporter group and BHQ2 fluorescence quenching group respectively at its 5' end and 3' end; the specificity of H7 subtype avian influenza virus HA gene The sex probe carrie...

Embodiment 2

[0077] Optimization of reaction system and program for triple fluorescence quantitative RT-PCR

[0078] The present invention optimizes the amount of each probe, the concentration of each primer and the reaction parameters (annealing temperature) in the reaction program of the triple fluorescence quantitative RT-PCR reaction system.

[0079] In this example, it is preferable to use the response surface optimization design method to systematically design and analyze the reaction system and reaction program, and construct the corresponding response surface to obtain the optimal area. In this example, MODDE 12.1 (Umetrics, Sweden) software was used to optimize the central composite surface design as the response surface model in the central composite design, and the steepest descent method was used to find the optimal area, and a total of 17 optimization experiments were designed (as shown in Table 3 ).

[0080] Table 3 Triple fluorescence quantitative RT-PCR system optimization...

Embodiment 3

[0090] Drawing of standard curve

[0091] Using 10-fold serial serial dilutions of H5, H7 and H9 standard strain genomic RNAs as templates, according to the optimized reaction system and program, perform triple fluorescent quantitative RT-PCR amplification, and set up 3 for each reaction Repeat, set positive and negative controls at the same time, respectively use the logarithm of the initial nucleic acid copy number of the standard RNA of H5, H7 and H9 strains as the X-axis, and the Ct value as the Y-axis to draw a standard curve, and quantify the unknown samples through the standard curve. The amplification efficiency of the reaction and the slope and correlation coefficient of the standard curve were obtained. The result is as figure 1 Shown (wherein A is the H5 amplification curve; B is the H5 standard curve; C is the H7 amplification curve; D is the H7 standard curve; E is the H9 amplification curve; F is the H9 standard curve),

[0092] The H5 standard curve equation o...

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Abstract

The invention discloses an avian influenza virus H5, H7 and H9 subtype triple fluorescent quantitative RT-PCR detection kit, and provides an optimal reaction system and reaction program of the kit anda use method thereof. The specificity of the kit reaches 100%, and the kit can specifically detect H5, H7 or H9 subtype AIV in the detection process of H1-H13 subtype AIV, NDV, IBV, IBDV and other common avian viruses. The detection sensitivity of the kit on the H5, H7 and H9 subtype avian influenza viruses in a sample can reach 50 copies / mu L, 50 copies / mu L and 50 copies / mu L. On the premise ofensuring single-tube multi-detection high detection efficiency and low cost, the kit has the characteristics of low cross contamination rate, simplicity and convenience in operation, fluorescence monitoring property, quickness, accuracy, high specificity, high sensitivity and high stability.

Description

technical field [0001] The invention belongs to the field of virus molecular biology detection, and in particular relates to a triple fluorescent quantitative RT-PCR detection kit for H5, H7 and H9 subtypes of avian influenza virus and its application. Background technique [0002] Avian influenza virus (avian influenza virus, AIV) is the pathogen of avian influenza (Avian influenza, AI). Negative strand RNA. The main avian influenza viruses prevalent in my country belong to H5, H7 and H9 subtypes. At present, all HPAIVs belong to H5 or H7 subtypes, and avian influenza caused by H5 and H7 subtypes is included in OIE as avian influenza that must be reported. Although the avian influenza virus of the H9 subtype belongs to LPAIV, it is currently considered to be highly pathogenic to poultry and cause serious damage to its production performance. In addition, H5, H7, and H9 subtypes are currently important subtypes of highly pathogenic avian influenza (referred to as human av...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166
Inventor 谭立恒闫丽萍古柏霖李源刘萌
Owner NANJING AGRICULTURAL UNIVERSITY
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