Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus

A porcine circovirus and real-time fluorescence technology, applied in the biological field, can solve the problems of inability to detect PCV2 and PCV1 by double real-time fluorescent PCR, single detection of PCV2, and easy occurrence of false positives, etc., so as to reduce the detection cost, high sensitivity and reliability. Good results

Active Publication Date: 2015-06-24
北京世纪元亨动物防疫技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these conventional methods are time-consuming, labor-intensive, low-sensitivity, and prone to false positives.
Among the current PCV detection methods, the established real-time fluorescent PCR detection method mainly for PCV2, or PCV1 real-time fluorescent PCR detection method, can only detect PCV2 or PCV1 alone, and cannot perform dual real-time fluorescent PCR detection on PCV2 and PCV1

Method used

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  • Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus
  • Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus
  • Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Screening of porcine circovirus type 1 and type 2 dual real-time fluorescent PCR method specific primers and probes

[0043] (1) Design of primers and probes for porcine circovirus

[0044] The specific primers for porcine circovirus PCV1 and PCV2 refer to: an oligonucleotide chain with a length of about 20 bases, a highly conserved specific nucleotide fragment of the porcine circovirus ORF2 gene;

[0045] The specific probes for porcine circovirus PCV1 and PCV2 refer to: an oligonucleotide chain with a length between 13 and 30 bases, whose 5' end is labeled with a fluorescent excitation group such as FAM or HEX, and the 3' The terminal label is a quencher group that does not emit light by itself.

[0046] According to the obtained ORF2 gene-specific highly conserved region, multiple real-time fluorescent PCR amplification primers and TaqMan probes for porcine circovirus PCV1 and PCV2 were designed using Primer Express3.0 software. For the specific sequences...

Embodiment 2

[0066] Example 2. Optimization of porcine circovirus type 1 real-time fluorescent PCR detection method

[0067] (1) Design of primers and probes for porcine circovirus type 1 real-time fluorescent PCR method

[0068] Refer to Example 1 for the primer and probe design of porcine circovirus type 1 real-time fluorescent PCR method, and see Table 4 for the specific sequence.

[0069] (2) Reaction system and conditions of porcine circovirus type 1 real-time fluorescent PCR method

[0070] The optimization principle of porcine circovirus type 1 real-time fluorescent PCR method is: by optimizing the following conditions, the same sample can obtain the maximum amplification efficiency and the minimum Ct value.

[0071] a. Determination of the optimal fluorescent primer concentration: the fluorescent primer concentration was screened between 300nM and 800nM.

[0072] b. Determination of the optimum probe concentration: the probe concentration is selected between 100nM and 500nM.

[...

Embodiment 3

[0090] Example 3. Optimization of porcine circovirus type 2 real-time fluorescent PCR detection method

[0091] (1) Design of primers and probes for porcine circovirus type 2 real-time fluorescent PCR method

[0092] Refer to Example 1 for the primer and probe design of porcine circovirus type 2 real-time fluorescent PCR method, and see Table 4 for the specific sequence.

[0093] (2) Reaction system and conditions of porcine circovirus type 2 real-time fluorescent PCR method

[0094] The optimization principle of porcine circovirus type 2 real-time fluorescent PCR method is: by optimizing the following conditions, the same sample can obtain the maximum amplification efficiency and the minimum Ct value.

[0095] a. Determination of the optimal fluorescent primer concentration: the fluorescent primer concentration was screened between 300nM and 800nM.

[0096] b. Determination of the optimum probe concentration: the probe concentration is selected between 100nM and 500nM.

[...

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Abstract

The invention discloses a dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, a kit and a detection method for type 1 and type 2 porcine circovirus. The dual real-time fluorescence PCR detection primer pair for type 1 and type 2 porcine circovirus is characterized in that the nucleotide sequence of the type 1 specific porcine circovirus is as shown in SEQ ID NO: 9 and SEQ ID NO: 11. The probe comprises the nucleotide sequence as shown in SEQ ID NO: 14. The nucleotide sequence of the type 2 specific porcine circovirus is as shown in SEQ ID NO: 2 and SEQ ID NO: 6. The probe comprises the nucleotide sequence as shown in SEQ ID NO: 7. The kit provided by the invention has the characteristics of rapidness, simpleness and convenience, strong specificity, high sensitivity and good reliability, and can be used for analyzing samples in batches at the same time, and differentiating whether the sample is type 1 porcine circovirus infection or type 2 porcine circovirus infection or not by reaction at one time, or common infection of type 1 and type 2 porcine circovirus infection, thereby, providing a powerful technical support for monitoring and preventing porcine circovirus epidemic situation. The kit has good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to dual real-time fluorescent PCR detection, in particular to primers, probes, a kit and a detection method for dual real-time fluorescent PCR detection of porcine circovirus type 1 and type 2. Background technique [0002] Porcine circovirus (porcine circovirus, PCV) belongs to Circoviridae, Circovirus genus. The virus is a small, non-encapsulated, icosahedral symmetrical, covalently closed, circular, single-stranded DNA virus with an average particle diameter of 17nm and replicates in a rolling circle. It is the smallest animal virus found so far . Brain, Allan et al. believed that PCV can be divided into two genotypes, PCV1 and PCV2, according to the pathogenicity, antigenicity and nucleotide sequence of PCV. At present, it is generally believed that PCV1 has no pathogenicity, including PCV-PK15 and other isolates that are prevalent in pigs but have no pathogenicity. Experiments have ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 孙明陈西钊乔明明陈南华
Owner 北京世纪元亨动物防疫技术有限公司
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