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, so as to reduce the detection cost, high sensitivity and reliability. Good results
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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.
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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 optimal probe concentration: the probe concentration is selected between 100nM and 500nM.
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