PCR (Polymerase Chain Reaction) primer for detecting and identifying porcine circovirus 3 (PCV3) and detection method and detection kit

The technology of a porcine circovirus and a kit, which is applied in the field of animal disease sanitation inspection, can solve the problems that there is no method or kit for detecting and identifying porcine circovirus type 3, so as to facilitate clinical detection and carry out epidemiological investigation, Great application prospect and high specific effect

Inactive Publication Date: 2017-08-04
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the research progress of PCV3 in my country, and there is no method or kit for detecting and identifying porcine circovirus type 3

Method used

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  • PCR (Polymerase Chain Reaction) primer for detecting and identifying porcine circovirus 3 (PCV3) and detection method and detection kit
  • PCR (Polymerase Chain Reaction) primer for detecting and identifying porcine circovirus 3 (PCV3) and detection method and detection kit
  • PCR (Polymerase Chain Reaction) primer for detecting and identifying porcine circovirus 3 (PCV3) and detection method and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Implementation example 1 The design of porcine circovirus type 3 PCR primers

[0047] 1. The present invention finds that porcine circovirus type 3 encodes two main open reading frames: rep and cap, both of which are in opposite directions on the DNA chain. By referring to and comparing all PCV3 cap gene sequences in GenBank, selecting a conserved nucleotide sequence as an amplification region, a pair of PCR primers for detecting porcine circovirus type 3 have been designed; the PCR primers are as follows:

[0048] Upstream primer PCV3-F: 5'-GGGCACACAGCCATAGAT-3';

[0049] Downstream primer PCV3-R: 5'-TTCCGGGACATAAATGCT-3'.

[0050] 2. After verification, the DNA of porcine circovirus type 3 was used as a template for amplification, and the results showed that the primer set could specifically amplify and detect porcine circovirus type 3.

Embodiment 2

[0051] Example 2 PCR primers for detection of porcine circovirus type 3 samples

[0052] 1. DNA extraction

[0053] (1) Take 30mg of swine disease tissue, cut the tissue into pieces as much as possible, and put them into 1.5ml centrifuge tubes.

[0054] (2) Add 450µL of lysis buffer (10mM Tris-HCl pH8.0; 100mM EDTA, pH8.0) to each tube.

[0055] (3) Add 50-100 µL of 10% SDS to each tube, and then add 2.5-5 µL of proteinase K (20 mg / ml) to make the final concentration reach 100 µg / mL, mix well, and incubate at 55°C for 3 hours to overnight (during this period, turn the tube upside down a few times) Second-rate).

[0056] (4) Add 150µL NaCl to the sample, centrifuge at 8000rpm for 20min at room temperature, remove the precipitate, then add an equal volume (500µL) of pre-cooled isopropanol to the supernatant, mix well, treat at 20°C for 30min, centrifuge at 14000rpm for 10min, remove the supernatant liquid. Wash the precipitate with 70% ALC and centrifuge to remove ALC, evapo...

Embodiment 3

[0072] Implementation example 3 The specific detection of porcine circovirus type 3 PCR primers

[0073] With reference to step 1 to step 3 in embodiment example 2, utilize the PCR primer described in embodiment 1 to porcine circovirus type 3, foot-and-mouth disease virus, porcine epidemic diarrhea virus, porcine senega valley virus, porcine deltacoronavirus, porcine Kub virus, porcine Boca virus and porcine Sapero virus were detected by PCR respectively. The virus strains were all donated by Guangdong Wen's Group.

[0074] Electrophoresis results such as figure 2 Shown, PCR primer of the present invention is positive reaction to porcine circovirus type 3, but to porcine vesicular virus, foot-and-mouth disease virus, porcine epidemic diarrhea virus, porcine senega valley virus, porcine deltacoronavirus, porcine Kubu Viruses, porcine Boca virus and porcine Sapero virus were negative, indicating that the primer has a strong specificity for porcine circovirus type 3.

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) primer for detecting and identifying porcine circovirus 3 (PCV3) and a detection method and a detection kit. A pair of PCR primers for detecting the PCV3 is designed by performing preference comparison with genomic sequences of the PCV3 according to a conserved sequence on a cap protein of the PCV3. Sequences of the forward and reverse primers PCV3-F/R of the PCR are sequentially shown as SEQ ID NO.1 to SEQ ID NO.2. The invention further provides a PCR detection method and kit for the PCV3. The primer and the detection method disclosed by the invention are capable of rapidly, conveniently and efficiently detecting and identifying the PCV3, the specificity is high, the sensitivity is high, and the detection method is simple and high-efficiency in operation, is convenient for clinical detection and convenient for developing epidemiological investigation and has great application prospects.

Description

technical field [0001] The invention belongs to the technical field of sanitation inspection of animal diseases, and in particular relates to a PCR primer, a detection method and a detection kit for detecting and identifying porcine circovirus type 3. Background technique [0002] Porcine circovirus belongs to the Circoviridae family and is the smallest virus among swine pathogens. Two types of circoviruses are known to infect pigs: porcine circovirus type 1 and porcine circovirus type 2. Among them, porcine circovirus type 2 (PCV2) infection can cause various clinical diseases, resulting in huge economic losses in the world pork industry, while porcine circovirus type 1 (PCV1) is not pathogenic. The incubation period after porcine circovirus infection is long, even if it is infected in the embryonic period or early after birth, clinical symptoms will appear one after another after weaning. Studies have found that PCV2 can cause the following diseases, including porcine mu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 蓝天马静云孙媛麦凯杰
Owner SOUTH CHINA AGRI UNIV
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