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Kit and method for detecting porcine circovirus type-2 (PCV-2) and subtypes thereof

A porcine circovirus and kit technology, which is applied in biochemical equipment and methods, material excitation analysis, and microbial determination/inspection, etc., can solve the problems of undetermined, laborious, and time-consuming mixed infection, and achieve easy and sensitive results. High performance, simple and quick effect

Inactive Publication Date: 2011-10-05
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional typing identification uses the sequencing method, which has certain limitations. It is impossible to determine the mixed infection of different subtypes of PCV-2 in the same pig, and it is time-consuming and laborious. Therefore, a fast, convenient and intuitive method should be established. Fluorescent PCR detection method, which can achieve the purpose of detection and genotyping

Method used

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  • Kit and method for detecting porcine circovirus type-2 (PCV-2) and subtypes thereof
  • Kit and method for detecting porcine circovirus type-2 (PCV-2) and subtypes thereof
  • Kit and method for detecting porcine circovirus type-2 (PCV-2) and subtypes thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 kit composition

[0061] Fluorescent PCR reaction solution 2 tubes (25μL / reaction×100), the final concentration of dATP, dTTP, dGTP and dCTP is 0.24mM, Mg 2+ The final concentration is 2.4mM, the final concentration of primers C1 and C2 is 0.32μM, 1 tube of probe a and b mixture (2μL / reaction×100), the concentration is 5μM, and 1 tube of hot-start Taq enzyme and enzyme protection agent mixture (2μL / reaction×100), wherein the concentration of hot-start Taq enzyme is 1U / μL, the concentration of enzyme protection agent is 0.2U / μL, 1 tube of positive plasmid control a and b (250 μL / tube), and 1 tube of negative plasmid control (250 μL / Tube).

[0062] This kit uses a 30 μL reaction system, and the reaction solution is composed of: 25 μL of fluorescent PCR reaction solution, 2 μL of enzyme, 1 μL of probe mixture and 2 μL of template.

Embodiment 2

[0063] The usage method of embodiment 2 porcine circovirus type 2 double fluorescent PCR detection kit

[0064] 1. Sample processing:

[0065] Please extract the DNA by yourself. The positive control does not need to be extracted, and the sample is added directly.

[0066] 2. Detection of fluorescent PCR

[0067] (1) Take the PCR reaction solution, hot-start Taq enzyme and probe according to the number of samples to be tested n (n=number of samples to be tested + 2), mix them in a centrifuge tube, vibrate on a vortex shaker, and pack in each tube , cover the tube and set aside.

[0068] (2) Now add the negative control solution into an aliquot tube, take the DNA of each sample and add it to the corresponding reaction tube; finally take out the positive control solution and add it to another reaction tube, mark each reaction tube and centrifuge, take it out and put it in a fluorescent PCR instrument .

[0069] (3) Fluorescence PCR reaction conditions: pre-denaturation at 94...

Embodiment 3

[0079] Embodiment 3 kit specificity test

[0080] Take 2 μL of DNA from 4 control samples including porcine circovirus type 1 strain, porcine pseudorabies virus strain, porcine parvovirus, and PK-15 cells as templates for specific PCR amplification of the kit, and set a negative control group at the same time.

[0081] PCR amplification conditions: pre-denaturation at 95°C for 3 minutes, cycled 40 times according to the following parameters: denaturation at 95°C for 15 sec, fluorescence collection at 58°C for 40 sec.

[0082] The results of fluorescent PCR showed that only the DNA of PCV-2a and PCV-2b amplified curves, while the DNA of porcine circovirus type 1 strain, porcine pseudorabies strain, porcine parvovirus, and PK-15 cells as control samples were all amplified. No such amplification curve appears (see Figure 4 ).

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Abstract

The invention provides a kit for detecting porcine circovirus type-2 (PCV-2) and subtypes thereof, belonging to the field of biological detection and solving the technical problems of complex process and long period of a method for detecting the PCV-2 and the subtypes thereof. The kit internally comprises fluorescent PCR (Polymerase Chain Reaction) reaction liquid, a TaqMan specific probe a and a TaqMan specific probe b, wherein the fluorescent PCR reaction liquid contains specific primers, the sequence of the upstream primers is as shown in SEQIDNO: 3, and the sequence of the downstream primers is as shown in SEQIDNO: 4; the TaqMan specific probe a aims at a PCV-2a (Porcine Circovirus-2a); and the TaqMan specific probe b aims at a PCV-2b (Porcine Circovirus-2b). The invention also provides a method for detecting the PCV-2 and the subtypes thereof by adopting the kit. The bifluorescence PCR detection kit can fast and accurately detect the PCV-2 and also identify the subtypes of the PCV-2 and is suitable for the fast diagnosis of the PCV-2, epidemiological survey, risk assessment and genotyping analysis.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a virus detection kit and a method thereof, in particular to a kit and a method for detecting porcine circovirus type 2 and typing. Background technique [0002] Porcine circovirus type 2 (Porcine circovirus-2, PCV-2) can cause multisystemic wasting syndrome (PMWS), porcine dermatitis and nephritic syndrome (PDNS), reproductive failure, and porcine respiratory disease syndrome in weaned piglets. PMWS syndrome (PRDC), proliferative and necrotizing pneumonia (PNP) and congenital tremor (CT) and other diseases, but PMWS is the most common, and the disease is widely prevalent in the world, with a mortality rate ranging from 10% to 30%. . Lang Hongwu et al. (Lang Hongwu, 2000) used the ELISA method to detect 559 serum samples from 22 pig herds in seven provinces (cities) including Beijing, Hebei, Shandong, Tianjin, Jilin and Henan. The total positive rate of the sampl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 刘健刘佩红周锦萍王建张维谊鞠厚斌葛菲菲
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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