PCV2-PRV (porcine circovirus type 2-porcine pseudorabies virus) two-temperature type double PCR (polymerase chain reaction) detection primers and diagnosis method thereof

[0030] PCV2-PRV two-temperature double PCR detection primers, including PCV2 virus primer pair and PRV virus primer pair;

[0031] Wherein, the PCV2 virus primer pair includes upstream primer PCV2-F1 and downstream primer PCV2-F2; the PRV virus primer pair includes upstream primer PRV-F3 and downstream primer PRV-F4;

[0032] The sequence of the upstream primer PCV2-F1 is: 5'-AGTCTCATCCACAGCTGATTC-3';

[0033] The sequence of the downstream primer PCV2-F2 is: 5'-CATCTTGGCCAGATCCTCCG-3';

[0034] The sequence of the upstream primer PRV-F3 is: 5'-TCCTGTACGCCCTATTCA-3';

[0035] The sequence of the downstream primer PRV-F4 is: 5'-CCGACTCTCGTGATGTCATCC-3'.

[0037] The PCV2-PRV two-temperature double PCR diagnostic method comprises the following steps:

[0038] 1) Synthesize PCV2 virus primers to PCV2-F1 / PCV2-F2 and PRV virus primers to PRV-F3 / PRV-F4;

[0039] 2) Extracting viral genomic DNA from PCV2 and PRV disease materials;

[0040] 3) PCV2 DNA and PRV DNA were amplified and detected by two-temperature PCR.

[0041] Further, the method for extracting the viral genomic DNA in step 2) is as follows: take 0.1 g of PCV2 and PRV positive disease materials respectively, place them in an Eppendorf tube, add 1 ml of TE buffer solution, and twist it into a homogenate with a twister, - Freeze and thaw three times at 70°C, centrifuge to get 0.2ml of the supernatant, then use a viral DNA extraction kit to extract viral DNA, and finally elute in 50ul TE buffer and store at -20°C to obtain PCV2DNA and PRV DNA.

[0042] Further, the method of the two-temperature type PCR amplification in step 3) is:

[0043] Use PCV2 and PRV DNA as templ

[0053] The method for determining the optimal annealing temperature for two-temperature PCR is: mix the 50 μl reaction system in Example 2 and put it in a PCR amplification instrument. The amplification conditions are: 95°C for 5 minutes; ℃, 46.9°C, 49.0°C, 51.4°C, 53.7°C, 56.3°C, 58.6°C, 61.0°C, 63.1°C, 64.4°C for 45s, 35 cycles; the final extension at 72°C for 10 min, after agarose gel electrophoresis, as figure 1 As shown, the results show that the optimal annealing temperature of PRV is 61°C, at which point the two specific bands can be clearly distinguished, and the product amplification efficiency is the highest.