Porcine parvovirus microdroplet number PCR absolute quantitative detection kit

A parvovirus and kit technology, which is applied in the detection/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inability to accurately quantify viral nucleic acids and inability to reflect the core concept of digital PCR very well, and achieve The effect of preventing large-scale outbreaks, high accuracy, and accurate and reliable results

Inactive Publication Date: 2017-12-08
CHINA ANIMAL DISEASE CONTROL CENT
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Each of the above methods can only achieve qualitative and semi-quantitative detection, and cannot accurately quantify viral nucleic acid, and there are still certain limitations in sensitivity and specificity.
[0004] The concept of digital PCR (Digital PCR, dPCR) was adopted and published by Bert Vogelstein as early as 1999, and its original intention was to obtain a large number of normal somatic cells from clinical samples (such as urine, lymph, plasma, stool, etc.) A small amount of mutant cells were detected in the sample, but because the consumables that could be used to dilute samples at that time were only 384-well plates, it could not reflect the core concept of digital PCR - "terminal dilution" (terminal dilution) very well.

Method used

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  • Porcine parvovirus microdroplet number PCR absolute quantitative detection kit
  • Porcine parvovirus microdroplet number PCR absolute quantitative detection kit
  • Porcine parvovirus microdroplet number PCR absolute quantitative detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, design and screening of primers and probes

[0064] 1. Design and screening of primers and probes

[0065] 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:

[0066] F1: 5'-CCCACTGATGACACCTTTG-3';

[0067] R1: 5'-AACAGAGGCTCATCCTGGT-3';

[0068] P1: 5'-CGCGTAGAACTGACAACAACGCTGA-3';

[0069] F2 (sequence 1 of the sequence listing): 5'-AGTGGGTCGGAGCACCAGTTC-3';

[0070] R2 (sequence 2 of the sequence listing): 5'-CGCAGACACGAATCCAGAGGCT-3';

[0071] P2 (sequence 3 of the sequence listing): 5'-AACTGTGACAACTGGAACGCTGACGCA-3'.

[0072] F1 and F2 are upstream primers, R1 and R2 are downstream primers, and P1 and P2 are probes. The 5' end of P1 is labeled with the fluorescent group FAM, and the 3' end is labeled with the fluorescent quencher group BHQ1. The 5' end of P2 is labeled with the fluorescent...

Embodiment 2

[0080] Embodiment 2, optimization of relevant reaction parameters in digital PCR

[0081] 1. Optimization of heating and cooling speed

[0082] 1. Take porcine parvovirus and extract genomic DNA.

[0083] 2. Using the genomic DNA obtained in step 1 as a template, digital PCR is performed using the primer probe set F2R2P2.

[0084](1) Prepare the following system (20 μL): 10 μL 2×ddPCR Supermix for Probes, 1.8 μL upstream primer, 1.8 μL downstream primer, 0.8 μL probe, 2 μL template, 3.6 μL RNase Free dH 2 O. In the system, the concentration of the upstream primer is 0.9 μM, the concentration of the downstream primer is 0.9 μM, and the concentration of the probe is 0.4 μM.

[0085] (2) Take the droplet generating card and fix it on the base, add 20 μL of the system prepared in step (1) to each well of the 8 holes in the middle row, add 70 μL of the microdroplet generating oil to each well of the 8 holes in the bottom row, Then place the base with the droplet generation card...

Embodiment 3

[0120] Embodiment 3, the preparation of kit

[0121] 1. Preparation of each reagent

[0122] Solution A is the one-step ddPCR probe master mix. The composition of each 900 μL solution A is as follows: 500 μL 2×ddPCRSupermix for Probes, 90 μL F2 solution (the concentration of F2 in the F2 solution is 10 μM), 90 μL R2 solution (the concentration of R2 in the R2 solution is 10 μM), 40 μL P2 solution (in the P2 solution The concentration of P2 is 10μM), 180μL RNase Free dH 2 O.

[0123] Solution B is droplet generating oil.

[0124] Solution C is a positive control. Preparation method of solution C: Genomic DNA of porcine parvovirus was extracted and diluted with Tris-EDTA buffer (pH 8.0, 0.01M) to make the concentration 10000 copies / microliter.

[0125] Solution D is a negative control. Solution D is RNase Free dH 2 O.

[0126] 2. Assembly of Microdroplet Digital PCR Absolute Quantitative Detection Kit

[0127] The composition of the kit is as follows: Solution A, Soluti...

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Abstract

The invention discloses a porcine parvovirus microdroplet number PCR absolute quantitative detection kit. A primer probe group provided by the invention consists of a primer F2 as shown in a sequence 1, a primer R2 as shown in a sequence 2 and a probe P2; the nucleotide sequence of the probe P2 is as shown in a sequence 3 in a sequence table; and one tail end of the probe is provided with a fluorescent report gene and the other tail end of the probe is provided with a fluorescent quenching group. The application of the primer probe group is as follows: identifying the porcine parvovirus; detecting whether a to-be-detected sample contains the porcine parvovirus or not; and detecting the content of the porcine parvovirus in the to-be-detected sample. The porcine parvovirus microdroplet number PCR absolute quantitative detection kit has important application value in prevention and control of the porcine parvovirus, and contributes to control epidemic situation from the source and effectively prevent large-scale outburst of porcine epidemic diseases.

Description

technical field [0001] The invention belongs to the field of virus detection, in particular to a porcine parvovirus droplet digital PCR absolute quantitative detection kit. Background technique [0002] Porcine parvovirus (PPV) was discovered by Mayr and Mahnl in 1966 when using porcine kidney primary cells for tissue culture of swine fever virus, and was subsequently identified as a DNA virus, one of the main pathogens causing reproductive disorders in sows , Cartwight et al first confirmed its pathogenicity. Since the mid-1960s, many countries in Europe, America, and Asia have isolated the virus or detected its antibody. In China, Pan Xuezhu et al. (1982) isolated the virus for the first time in Shanghai, and since then there have been reports of PPV isolation in different provinces across the country. It is characterized in that sows are infected in the early stage of pregnancy, and the embryo or fetus is invaded through the placenta, causing abortion, embryo death, fet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/159
Inventor 原霖杨林宋晓晖王传彬韩焘吴佳俊亢文华倪建强周智訾占超王晓英毕一鸣王静汪葆玥陈亚娜
Owner CHINA ANIMAL DISEASE CONTROL CENT
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