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Porcine circovirus type 3 PCR detection primer pair and kit

A porcine circovirus and detection primer technology, applied in the biological field, can solve the problem of blank disease monitoring and achieve the effect of good detection specificity and high sensitivity

Inactive Publication Date: 2017-12-08
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no detection method for porcine circovirus

Method used

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  • Porcine circovirus type 3 PCR detection primer pair and kit
  • Porcine circovirus type 3 PCR detection primer pair and kit
  • Porcine circovirus type 3 PCR detection primer pair and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: The PCR detection method establishment of porcine circovirus type 3

[0045] 1. Primer design and synthesis

[0046]According to the ORF2 conserved region of the PCV-3 (KX966193) PCV3-US / SD2016 strain registered in GenBank, a pair of specific primers were designed using Primer Premier 5.0 software. The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Company. The sequence of the pair is shown in Table 1 (wherein, "F" represents the forward primer, and "R" represents the reverse primer):

[0047] Table 1: Primer information

[0048]

[0049] 2. Viral DNA extraction

[0050] Take the porcine circovirus type 3 virus liquid, and operate according to the operation manual of the MiniBEST Viral RNA / DNAExtraction Kit Ver.5.0 virus extraction kit from TaKaRa Company, the operation steps are as follows:

[0051] (1) Take 200 μL of virus liquid, 200 μL of Buffer VGB, 20 μL of proteinase K and 1 μL of Carrier RNA, mix them evenly, an...

Embodiment 2

[0092] Embodiment 2: PCR assay method optimization of porcine circovirus type 3

[0093] The reaction was optimized using a temperature gradient of 48°C, 50°C, 52°C, and 54°C. By optimizing the reaction conditions, it was confirmed that the following reaction system and amplification program were the best conditions:

[0094] Optimal reaction program: 25μL reaction system, including:

[0095] (1) 1 μL of the forward primer of the nucleotide sequence shown in 10 μmol / L SEQ ID NO:1; and 1 μL of the reverse primer of the nucleotide sequence shown in 10 μmol / L SEQ ID NO:2;

[0096] (2) 2.5 μL DNA template of the sample to be tested;

[0097] (3) Premix Taq premix 12.5μL;

[0098] (4)ddH 2 O 8 μL.

[0099] The optimal amplification program is:

[0100] (1) Pre-denaturation at 94°C for 5 minutes;

[0101] (2) Denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 35s, 38 cycles;

[0102] (3) Extend at 72°C for 7 minutes.

Embodiment 3

[0103] Embodiment 3: the kit preparation of porcine circovirus type 3 PCR detection

[0104] The porcine circovirus type 3 nucleic acid detection kit provided by the invention comprises: Premix Taq premix, ddH 2 O, the forward primer of the nucleotide sequence shown in SEQ ID NO:1, the reverse primer of the nucleotide sequence shown in SEQ ID NO:2, negative quality control product (ddH 2 O), positive quality control (prepared standard plasmid).

[0105] The reagents used in the preparation of this kit were mainly purchased from Takara Bioengineering (Dalian) Co., Ltd. (Takara), and the rest were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. and Beijing Chemical Plant. The preparation process of the kit is as follows:

[0106] 1. Preparation of PCR reaction solution

[0107] (1) Primer design and synthesis

[0108] According to the PCV3 ORF2 gene sequence published in GenBank, the sequence alignment was performed with MEGA 6.0 software to find out the co...

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Abstract

The invention discloses a porcine circovirus type 3 PCR detection primer pair and a porcine circovirus type 3 PCR detection kit. The primer pair includes a forward primer having a nucleotide sequence shown as SEQ ID NO:1 and a reverse primer having a nucleotide sequence shown as SEQ ID NO:2. The primer pair is used for amplifying an ORF2 gene segment of porcine circovirus type 3, and a target gene segment, which is amplified, is 408bp. The invention also provides the porcine circovirus type 3 PCR detection kit and a using method of the kit. The primer pair provided by the invention, when used for detecting the porcine circovirus type 3, has the characteristics of being high in accuracy, strong in specificity, high in sensitivity, simple, rapid and the like; and the primer pair is applicable to etiological investigation of the porcine circovirus type 3.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method for porcine circovirus type 3, in particular to a PCR detection primer pair and a kit for porcine circovirus type 3. Background technique [0002] Porcine circovirus (PCV) is a member of the genus Circovirus in the family Circoviridae. PCV was first discovered by German scientists in 1974. In 1982, Tischer et al. used ultracentrifugation to purify the virus. After electron microscope observation and nucleic acid identification, it was determined that the diameter of the virus was about 14-17 nm, and its genome was a single-stranded circular DNA. Since the discovery of multisystem wasting syndrome in weaned piglets in Canada in 1991, relevant reports have been reported in many western developed countries. In 2002, porcine circovirus type 2 (PCV-2) caused an outbreak of multisystemic wasting syndrome in weaned piglets in most areas of my country. [0003] Porcine ci...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q2600/166
Inventor 张帆帆唐玉新叶昱宋德平李凯郭楠楠张敏吴琼黄冬艳
Owner JIANGXI AGRICULTURAL UNIVERSITY
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