Bird flu and newcastle disease double-droplet digital PCR absolute quantitative detection kit

A kit, avian influenza virus technology, applied in the determination/inspection of microorganisms, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of low sensitivity, unable to reflect the real situation of virus infection, poor repeatability, etc.

Inactive Publication Date: 2017-12-08
CHINA ANIMAL DISEASE CONTROL CENT
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Traditional detection methods, such as virus neutralization test (VNT), immunofluorescence, immunoelectron microscopy, enzyme-linked immunosorbent assay, etc., take a long time to exist, rely on experienced testing personnel, complex test operations, and involve live virus operations , high requirements for the detection environment, and many shortcomings such as low sensitivity and poor repeatability
Although nucleic acid detection methods such as RT-PCR have the advantages of stronger specificity, higher sensitivity, higher degree of automation, and better repeatability of results than traditional methods, they can only achieve qualitative and semi-quantitative detection of avian influenza and Newcastle disease. detection, it is impossible to perform accurate absolute quantitative detection of avian influenza and Newcastle disease nucleic acid, so the detection resu

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1, design and screening of primers and probes

[0081] 1. Design and screening of primers and probes for detection of avian influenza virus

[0082] Through a large number of sequence acquisition, analysis, comparison and preliminary experiments, four primers and two probes were designed and preliminarily screened. The nucleotide sequences are as follows:

[0083] AIV-F1 (sequence 1 of the sequence listing): 5'-ACYGAGGTCGARACGTACGT-3';

[0084] AIV-R1 (sequence 2 of the sequence listing): 5'-CTTCAAGTYTCTGCGCGATCT-3';

[0085] AIV-P1 (Sequence 3 of the Sequence Listing): 5'-CTCTCTATCATCHCGTCAGGCMC;

[0086] AIV-F2: 5'-TAACCGAGGTCGAMACGTACGT-3';

[0087] AIV-R2: 5'-CGCAGARACTTGAAGATGTCTTTG-3';

[0088] AIV-P2: 5'-TATCATCSCGTCAGGCVCCTCAA;

[0089] In the above nucleotide sequence, Y represents C or T, R represents A or G, H represents A, T or C, M represents A or C, S represents G or C, and V represents G, A or C.

[0090] Both AIV-F1 and AIV-F2 are upstre...

Embodiment 2

[0115] Embodiment 2, optimization of relevant reaction parameters in digital RT-PCR

[0116] 1. Optimization of annealing temperature

[0117] 1. Take avian influenza virus and extract total RNA.

[0118] 2. Take Newcastle disease virus and extract total RNA.

[0119] 3. Mix the total RNA obtained in step 1 with the total RNA obtained in step 2 to obtain an RNA mixture.

[0120] 4. Using the RNA mixture obtained in step 3 as a template, digital RT-PCR was performed using a primer combination consisting of the primer probe set AIV-F1R1P1 and the primer probe set NDV-F1R1P1.

[0121](1) Prepare the following system (20 μL): 10 μL 2×One-step RT-ddPCR Supermix for probes, 1 μL AIV-F1, 1 μL AIV-R1, 0.2 μL AIV-P1, 1 μL NDV-F1, 1 μL NDV-R1, 0.2 μL NDV-P1, 2 μL template, 3.6 μL RNase Free dH 2 O. In the system, the concentration of the upstream primer is 0.5 μM, the concentration of the downstream primer is 0.5 μM, and the concentration of the probe is 0.1 μM.

[0122] (2) Take ...

Embodiment 3

[0147] Embodiment 3, the preparation of kit

[0148] 1. Preparation of each reagent

[0149] Solution A is the one-step ddPCR probe master mix. The composition of each 900 μL solution A is as follows: 500 μL 2×One-step RT-ddPCR Supermix for probes, 90 μL AIV-F1 solution (the concentration of AIV-F1 in the AIV-F1 solution is 10 μM), 90 μL AIV-R1 solution (AIV-R1 The concentration of AIV-R1 in the solution is 10 μM), 20 μL of IV-P1 solution (the concentration of AIV-P1 in the AIV-P1 solution is 10 μM), 90 μL of NDV-F1 solution (the concentration of NDV-F1 in the NDV-F1 solution is 10 μM) , 90 μL NDV-R1 solution (the concentration of NDV-R1 in the NDV-R1 solution is 10 μM), 20 μL NDV-P1 solution (the concentration of NDV-P1 in the NDV-P1 solution is 10 μM).

[0150] Solution B is droplet generating oil.

[0151] Solution C is a positive control. The preparation method of solution C: extract the total RNA of avian influenza virus and Newcastle disease virus respectively, mix t...

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Abstract

The invention discloses a bird flu and newcastle disease double-droplet digital PCR absolute quantitative detection kit. A primer probe combination is provided and consists of a primer probe set AIV-F1R1P1 and a primer probe set NDV-F1R1P1; the primer probe set AIV-F1R1P1 consists of a primer AIV-F1, a primer AIV-R1 and a probe AIV-P1; the primer AIV-F1 is as shown in the sequence 1, and the primer AIV-R1 is as show in the sequence 2; the nucleotide sequence of the probe AIV-P1 is the sequence 3; the primer probe set NDV-F1R1P1 consists of a primer NDV-F1, a primer NDV-R1 and a probe NDV-P1; and the primer NDV-F1 is as shown in the sequence 4, the primer NDV-R1 is as shown in the sequence 5, and the nucleotide sequence of the probe NDV-P1 is the sequence 6. The bird flu and newcastle disease double-droplet digital PCR absolute quantitative detection kit has a significant application value on prevention and control of bird flu virus and/or newcastle disease virus, and contributes to controlling epidemic situations from the source and effectively preventing large-scale outbreak of bird epidemic diseases.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a double droplet digital PCR absolute quantitative detection kit for bird flu and Newcastle disease. Background technique [0002] Avian influenza virus (AIV) is a type A influenza virus. Influenza viruses belong to the Orthomyxoviridae family of RNA viruses and are divided into three types: A, B, and C. Among them, influenza A virus is mainly found in poultry, and it is an infectious disease caused by influenza A virus in poultry with various symptoms ranging from respiratory system to severe systemic sepsis. The International Veterinary Bureau defines it as a Class A infectious disease. In 1994, 1997, 1999, and 2003, bird flu broke out in Australia, Italy, Hong Kong, and the Netherlands, respectively. In 2005, it broke out mainly in Southeast Asia and Europe. It is mainly transmitted through the respiratory tract, through close contact with infected poultry and thei...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/159C12Q2537/143C12Q2521/107
Inventor 原霖吴佳俊王传彬宋晓晖訾占超杨林亢文华倪建强周智韩焘王晓英毕一鸣王静汪葆玥陈亚娜
Owner CHINA ANIMAL DISEASE CONTROL CENT
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