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Porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection primer pair and kit

A real-time fluorescent quantitative, porcine circovirus technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of low sensitivity, inability to quantify virus content, etc. , the detection specificity is good

Inactive Publication Date: 2017-11-10
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the conventional PCR detection method is widely used to detect PCV-3, but the sensitivity of ordinary PCR detection is relatively low, and it cannot quantify the virus content and other disadvantages, and clinically, the disease of pigs has a certain positive correlation with the virus content

Method used

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  • Porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection primer pair and kit
  • Porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection primer pair and kit
  • Porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection primer pair and kit

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Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Establishment of SYBR Green I real-time fluorescent quantitative PCR detection method for 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] Extract viral RNA / DNA according to the operation manual of MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 Viral Extraction Kit from TaKaRa Company, the operation steps are as follows:

[0051] (1) After the grinder grinds the disease material, centrifuge to get the supernatant;

[0052] (2) Mix 200 μL supern...

Embodiment 2

[0096] Embodiment 2: SYBR Green I real-time fluorescent quantitative PCR assay method optimization of porcine circovirus type 3

[0097] Use temperature gradients of 48°C, 50°C, 52°C, and 54°C to optimize the reaction. By optimizing the reaction conditions, it is confirmed that the following reaction system and amplification program are the best conditions:

[0098] Optimal reaction program: 20μL reaction system, including:

[0099] (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;

[0100] (2) 2 μL of DNA template of the sample to be tested;

[0101] (3) 10 μL of SYBR Premix Ex Taq premix;

[0102] (4)ddH 2 O 6 μL.

[0103] The optimal amplification procedure is:

[0104] (1) Pre-denaturation at 95°C for 90s;

[0105] (2) Denaturation at 94°C for 5s, annealing at 58°C for 30s, 40 cycles;

[0106](3) Denaturation at 95°C for 15s, extension at...

Embodiment 3

[0107] Embodiment 3: The kit preparation of porcine circovirus type 3 SYBR Green I real-time fluorescent quantitative PCR detection

[0108] The porcine circovirus type 3 nucleic acid detection kit provided by the invention comprises: SYBR Premix Ex 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).

[0109] 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:

[0110] 1. Preparation of PCR reaction solution

[0111] (1) Primer design and synthesis

[0112] According to the PCV-3 ORF2 gene sequence published in GenBank, the sequence alignmen...

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Abstract

The invention discloses a porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection primer pair and a kit. The primer pair comprises a positive primer of nucleotide sequence as shown in SEQ ID NO:1 and a negative primer of nucleotide sequence as shown in SEQ ID NO:2. The primer pair is used for amplifying a partial segment of an ORF2 gene of the porcine circovirus type III, and the size of the amplified target gene segment is 131 bp. The invention also provides a porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection kit and a use method of the kit. When the primer pair is used for detecting the porcine circovirus type III, the primer pair has the characteristics of high specificity, high sensitivity, high repeatability, rapidness and the like, and can be applied to etiology detection of the porcine circovirus type III.

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 SYBR Green I real-time fluorescence quantitative PCR detection primer pair and a kit for porcine circovirus type 3. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is the smallest known virus capable of self-replication in mammalian cells. Enveloped, single-stranded circular DNA virus, mainly infects monocyte / macrophage lineage cells, among which porcine alveolar macrophages are the main target cells. According to the antigenicity, pathogenicity and nucleotide sequence differences of PCV, PCV is divided into three genotypes, PCV-1, PCV-2 and PCV-3, among which PCV-1 is not pathogenic, but widely exists in Pig body and pig-derived cell lines; PCV-2 is pathogenic and can cause a variety of diseases in pigs, including multisystemic wasting syndrome, porcine dermatitis and nephrotic syndrome, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 叶昱唐玉新张帆帆宋德平李凯郭楠楠张敏吴琼黄冬艳
Owner JIANGXI AGRICULTURAL UNIVERSITY
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