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Primers, probe, kit and method used for detecting porcine circovirus 3 based on digital PCR technology

A porcine circovirus, technical detection technology, applied in the field of detection of porcine circovirus type 3 primers, probes and kits, to achieve the effects of avoiding economic losses, accurate virus content, broad application prospects and industrialization prospects

Inactive Publication Date: 2019-01-22
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing technology does not utilize digital PCR technology to carry out relevant research on the absolute quantitative detection of porcine circovirus type 3, whether it is feasible, related solutions, and a finished kit suitable for industrialization.

Method used

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  • Primers, probe, kit and method used for detecting porcine circovirus 3 based on digital PCR technology
  • Primers, probe, kit and method used for detecting porcine circovirus 3 based on digital PCR technology
  • Primers, probe, kit and method used for detecting porcine circovirus 3 based on digital PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Establishment of a kit for the absolute quantitative detection of porcine circovirus type 3 based on digital PCR technology

[0048] A kit for the absolute quantitative detection of porcine circovirus type 3 based on digital PCR technology, including a primer set, 2×ddPCR Supermix, RNase-free ultrapure water, virus total DNA extraction reagents, positive control and negative control.

[0049] (1) Design of primers for digital PCR amplification: The primers were designed with the specific conserved sequence of porcine circovirus type 3 as the target gene. The primer sequences are listed in Table 1.

[0050] Table 1 Primer sequence list

[0051]

[0052] (2) 2×ddPCR Supermix contains: 2×PCR Buffer, 2×Taq DNA Polymerase, 16μM FP, 16μM BP primer, 20μM probe, 400μM dNTP mix and 2×RNase Cocktail.

[0053] (3) The positive control is porcine circovirus type 3 DNA, and the negative control is ultrapure water.

[0054] (4) Bacterial total DNA extraction reagents ...

Embodiment 2

[0055] Embodiment 2 Absolute Quantitative Detection Method of Porcine Circovirus Type 3 Based on Digital PCR Technology

[0056] The method that utilizes the kit of embodiment 1 to detect porcine circovirus type 3 may further comprise the steps:

[0057] (1) Extraction of viral DNA:

[0058] 1) Take 1 g of the sample to be tested, add 600 μL of lysate A to grind, vortex and mix for 20 seconds, and let stand at room temperature for 10 minutes;

[0059] 2) Transfer the mixture to an adsorption column and centrifuge at 12,000×g for 30-60 seconds;

[0060] 3) Discard the liquid in the collection tube, add 500 μL of washing solution B to the adsorption column, and centrifuge at 12,000×g for 30-60 seconds;

[0061] 4) Discard the liquid in the collection tube, add 500 μL washing solution C to the adsorption column, and centrifuge at 12,000×g for 30-60 seconds;

[0062] 5) Discard the liquid in the collection tube, and centrifuge at 12,000×g for 2 minutes to dry the column;

[00...

Embodiment 3

[0081] Example 3 specificity verification

[0082] Use the kit of the present invention to detect clinically obtained PCV1, PCV2, porcine respiratory tract and reproductive syndrome virus, actinobacillus pleuropneumoniae, porcine pseudorabies virus, porcine parvovirus, classic porcine fever virus, and healthy pigs Serum samples, plus positive and negative control samples, a total of 10 samples, all samples have been verified by sequencing method, the test results are shown in figure 1 .

[0083] The experimental results show that after digital PCR amplification of positive samples, positive droplets appear above the threshold line, which means amplification. The template DNA extracted from serum samples of PCV1, PCV2, porcine respiratory and reproductive syndrome virus, porcine infectious pleuropneumoniae, porcine pseudorabies virus, porcine parvovirus, classic porcine fever virus, and healthy pigs was subjected to digital PCR After amplification, the fluorescence signal val...

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Abstract

The invention discloses primers, a probe, a kit and a method used for detecting porcine circovirus 3 based on the digital PCR technology. The primers and the probe comprise an upstream primer FP, a downstream primer BP and the probe Probe; and the detection kit comprises 2XddPCR Supermix, ultrapure water without RNA anzyme, a virus total DNA extracting reagent, a negative control and a positive control. According to the detection method, DNA of a to-be-detected sample is extracted, quantitative detection is carried out on the DNA of the to-be-detected sample by adopting the microdrop digital PCR technology, whether the to-be-detected sample contains the porcine circovirus 3 or not is judged through analysis on the obtained copy number after amplification, and the content of the porcine circovirus 3 is also determined. The primers, the probe, the kit and the method have the advantages that the operation is accurate and sensitive, the applicability is wide, the dependency on a standard curve is eliminated, the absolute quantification can be realized, the early discovery, early treatment and early prevention for the porcine circovirus 3 virus disease diagnosis can be realized, and thus the outbreak of diseases can be effectively controlled.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a primer, a probe, a kit and a method for detecting porcine circovirus type 3 based on digital PCR technology. Background technique [0002] Porcine circovirus (PCV) is a small, non-enveloped, single-stranded circular DNA virus belonging to the family Circoviridae. There are three main genotypes of porcine circovirus, namely PCV1, PCV2 and PCV3. Among them, PCV1 is not pathogenic, while PCV2 is pathogenic and is associated with various diseases of pigs, such as multisystem failure syndrome, dermatitis, nephrotic syndrome and congenital tremor in weaned piglets. PCV3 is a virus first discovered in the United States in 2016, which can cause pig reproductive failure and severe loss of newborn piglets and other symptoms. Pathogens have been detected in farms such as Liaoning, Jiangxi, and Chongqing in my country. At present, porcine circovirus has spread all over the pig-r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/70C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/159
Inventor 李丽丽温雯孟赫诚石磊陈洵
Owner JINAN UNIVERSITY
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