Multiple real time fluorescence quantifying PCR method for detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and classical swine fever virus
A technology of porcine pseudorabies virus and porcine circovirus, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as hazards and cross-contamination, and achieve good application stability and low cost.
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Embodiment 1
[0039] Example 1 Standard plasmid preparation
[0040] 1 Preparation of target fragments Extract nucleic acids of PRV, PPV, PCV-2, and HCV according to conventional methods, and store the nucleic acids at -20°C for later use. To design primers, use OLIGO6.24, compare the nucleic acid sequences in GENEBANK, and design primers (Table 1). The extracted PRV, PPV, PCV-2, and HCV viral nucleic acids were used as templates, and the primers in Table 1 were used for PCR amplification. The length of the amplified products was expected to be 265bp, 764bp, 792bp, and 303bp. The amplified products obtained were subjected to agarose gel electrophoresis, and the results were shown in figure 1 .
[0041] Table 1 Sequences of primer pairs for amplifying target genes
[0042]
[0043] 2 Preparation of standard plasmids Recover the target fragments according to the Plasmid Miniprep Kit operation method, connect the recovered products to the pMD18-T vector, transform Escherichia coli DH5α ...
Embodiment 2
[0045] Example 2 Establishment of SyMuReTi-PCR detection method for 4 kinds of viruses
[0046] 1.1 The dilution of the four virus standard plasmids is 4.97×10 using the Eppendorf Biophotometer to quantify the standard plasmids 12 Copies / μl, 3.44×10 12 Copies / μl, 8.18×10 12 Copies / μl, 7.98×10 12 Copies / μl, use EASY Dilution to dilute the standard plasmid at 1.0×10 8 ~1.0×10 1 Copies / μl 10-fold dilution, stored at -20°C.
[0047] 1.2 Detection primer design The target gene cloned in Example 1 was sequenced, and according to the determined sequence, four pairs of specific primers were designed using OLIGO6.24 (Table 2).
[0048] Table 2 Sequences of primer pairs for amplifying target genes
[0049]
[0050] 1.3 Add SyMuReTi-PCR to an Axygen Scientific Quality centrifuge tube (200 μl) in sequence: 9.5 μl of sterilized deionized water, 1 μl of dimethyl sulfoxide, 0.5 μl of each of the 4 pairs of primers in Table 2, and add different dilutions to different centrifuge tubes...
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