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Fluorescent quantitative PCR detection method and kit for porcine circovirus type II

A porcine circovirus and detection kit technology, which is applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc. Good repeatability and stability

Pending Publication Date: 2022-02-08
SHANGHAI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the fluorescently labeled nucleic acid sequence in the solution is complementary to the nucleic acid probe at the corresponding position on the gene chip, a set of completely complementary probe sequences can be obtained by determining the probe position with the strongest fluorescence intensity. The limitations are: Low detection rate of mosaicism or some special variants

Method used

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  • Fluorescent quantitative PCR detection method and kit for porcine circovirus type II
  • Fluorescent quantitative PCR detection method and kit for porcine circovirus type II
  • Fluorescent quantitative PCR detection method and kit for porcine circovirus type II

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of PCV2 Amplified Product Recombinant Plasmid and Standard Item

[0050] Utilize NCBI database to find out the ORF2 gene of PCV2, take PCV2 (BJ0804 strain) ORF2 gene as the target sequence, GenBank accession number is EU921257.1, full-length 149bp, specific sequence is as shown in SEQ ID NO.1, carry out design primer and probe The needles were designed using the software Primer Premier 5.0 for primers and probes, and Oligo DNA man 4.0 and PrimerAnalysis were used for comparison analysis. The specific sequences are as follows:

[0051] PCV2-P1: 5'-CGGATATTGTATTCCTGGTCGTA-3';

[0052] PCV2-P2: 5'-CCTGTCCTAGATTCCACTATTGATT-3';

[0053] PCV2-Probe: FAM-5'-CTAGGCCTACGTGGTCTACATTTC-3'-TAMRA.

[0054] Viral genomic DNA of PCV2 was extracted using the viral genomic DNA / RNA extraction kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0055] Porcine circovirus type 2 strain BJ0804 was used as a standard template, primers P1 and P2 were ...

Embodiment 2

[0060] Embodiment 2 A kind of fluorescence quantitative real-time detection method of porcine circovirus type 2 (PCV2)

[0061] 1. Design primers

[0062] Use the NCBI database to find out the ORF2 gene of PCV2. Based on the PCV2 (BJ0804 strain) ORF2 gene as the target sequence, the GenBank accession number is EU921257.1, the full length is 149bp, and the specific sequence is shown in SEQ ID NO.1. Needle, using the software Primer Premier 5.0 to design primers and probes, Oligo DNA man 4.0 and Primer Analysis for comparison analysis, the specific sequence is as follows:

[0063] PCV2-P1: 5'-CGGATATTGTATTCCTGGTCGTA-3';

[0064] PCV2-P2: 5'-CCTGTCCTAGATTCCACTATTGATT-3';

[0065] PCV2-Probe: FAM-5'-CTAGGCCTACGTGGTCTACATTTC-3'-TAMRA.

[0066] 2. Preparation of reaction system

[0067] In order to obtain an efficient and sensitive quantitative PCR reaction system, different primers, probe (or dye) concentrations, Mg 2+ Concentration or annealing temperature is optimized, and t...

Embodiment 3

[0080] Fluorescence quantitative PCR detection method specific verification of embodiment 3PCV2

[0081] According to the method of Example 2, the cDNA of PCV2 plasmid, PCV2 virus sample, classical swine fever virus (CSFV), porcine respiratory and reproductive disorder syndrome virus (PRRSV), porcine pseudorabies virus (PRV) is respectively used as template and double distilled water to carry out Fluorescent quantitative PCR detection was used to determine the specificity of the detection method.

[0082] Test results such as Figure 4 As shown, the abscissa is the cycle number, and the ordinate is the fluorescence value, 1-3: 3 repeated experiments of PCV2 plasmid, 4-6: 3 repeated experiments of PCV2 virus samples, 7: 3 repeated experiments of CSFV virus samples, 8: 3 repeated experiments for PRRSV virus samples, 3 repeated experiments for 9: PRV virus samples, 3 repeated experiments for 10: negative control.

[0083] Depend on Figure 4 It can be seen that the primers P1,...

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Abstract

According to the fluorescent quantitative PCR detection method and kit for the porcine circovirus type II, corresponding specific primers and probes are designed for the ORF2 gene of the porcine circovirus type 2, namely the PCV2, cross reaction with PCV1, CSFV, PRRSV and PRV is avoided, and the detection limit and the quantitative limit are 10 copy numbers and 100 copy numbers respectively. The kit disclosed by the invention is high in sensitivity, strong in specificity and good in stability, a circulation threshold (Ct) and a standard DNA template have an excellent linear relationship in a concentration range of 102-106 copies / mu L, and a correlation coefficient is 1; the invention is simple in operation and good in repeatability, the variation coefficient of the Ct value in an intra-batch experiment ranges from 0.59% to 1.05%, the variation coefficient of the Ct value in an inter-batch experiment ranges from 1.9% to 4.2%, and a simple, convenient and rapid diagnosis method is provided for detection of the porcine circovirus type II and investigation of epidemiology.

Description

technical field [0001] The invention belongs to the technical field of porcine circovirus detection, and in particular relates to a fluorescent quantitative PCR detection method and kit for porcine circovirus type 2. Background technique [0002] Porcine circovirus (Porcine Circovirus Type, PCV) is a member of the family Circoviridae and the genus Circovirus (Circovirus genus), and has two serotypes, namely, porcine circovirus type 1 (PCV1) and porcine circovirus Cyclovirus type 2 (PCV2). It is generally believed that PCV1 is not pathogenic, while PCV2 is pathogenic, and PCV2 is the main pathogen that causes multisystemic wasting syndrome (PMWS) in weaned piglets. [0003] PCV2 mainly destroys the pig's immune system, causing the body's immunosuppression and reduced resistance, leading to the secondary infection of other pathogenic microorganisms in the infected pigs. PCV2 is also closely related to various other clinical diseases of pigs. Since the first report of PMWS i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/166C12Q2531/113C12Q2545/114C12Q2563/107C12Q2545/113
Inventor 赵凯杨丹张晓霞张琪张皖静浦心袆赵斌安赵悦琪钱忠辉张俊平丁卫星张春玲赵本进王瑞阳张华弟
Owner SHANGHAI ACAD OF AGRI SCI
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