Multiple nested PCR (polymerase chain reaction) detection primer, kit and method for Neospora caninum, Brucella abortus and infectious bovine rhinotracheitis virus

[0081] Neospora, Brucella and bovine infectious rhinotracheitis virus multiplex nested PCR detection primers in this embodiment include: outer primer set:

[0082] Primer NC EX 1: 5'-CTTTTCAACCCTCAACCTTT-3';

[0083] Primer NC EX 2: 5'-TGACACCCAATCATACTGCTC-3';

[0084] Primer BA EX 1: 5'-CTACGGAATAACTCAGGGAAAC-3';

[0085] Primer BA EX 2: 5'-AACCCAACATCTCACGACAC-3';

[0086] Primer IBRV EX 1: 5'-ACGGACCTGGTGGACAAGAA-3';

[0087] Primer IBRV EX 2: 5'-GTGCGTGCCGTTGTAGCG-3';

[0088] Internal primer set:

[0089] Primer NC IN 3: 5'-TGAGTTGTATCGCCTTCTT-3';

[0090] Primer NC IN 4: 5'-ATTCACATTGCGTTTCG-3';

[0091] Primer BA IN 3: 5'-GGTAAAGGCTCACCAAGGC-3';

[0092] Primer BA IN 4: 5'-CAGGCGGAATGTTTAATGC-3';

[0093] Primer IBRV IN 3: 5'-AGGTGGTGGCCTTTGACC-3';

[0094] Primer IBRV IN 4: 5'-TCGTCTCGCAGCATTTCGTC-3'.

[0096] In this embodiment, Neospora, Brucella and bovine infectious rhinotracheitis virus multiple nested PCR detection kits include: outer primer set, inner primer set, positive control template, negative control template, TranStart Taq DNA polymerase, 10×TranStart Taq Buffer, dNTPs.

[0097] The outer primer set is:

[0098] Primer NC EX 1: 5'-CTTTTCAACCCTCAACCTTT-3';

[0099] Primer NC EX 2: 5'-TGACACCCAATCATACTGCTC-3';

[0100] Primer BA EX 1: 5'-CTACGGAATAACTCAGGGAAAC-3';

[0101] Primer BA EX 2: 5'-AACCCAACATCTCACGACAC-3';

[0102] Primer IBRV EX 1: 5'-ACGGACCTGGTGGACAAGAA-3';

[0103] Primer IBRV EX 2: 5'-GTGCGTGCCGTTGTAGCG-3';

[0104] The inner primer set is:

[0105] Primer NC IN 3: 5'-TGAGTTGTATCGCCTTCTT-3';

[0106] Primer NC IN 4: 5'-ATTCACATTGCGTTTCG-3';

[0107] Primer BA IN 3: 5'-GGTAAAGGCTCACCAAGGC-3';

[0108] Primer BA IN 4: 5'-CAGGCGGAATGTTTAATGC-3';

[0109] Primer IBRV IN 3: 5'-AGGTGGTGGCCTTTGACC-3';

[0110] Primer IBRV IN 4: 5'-TCGTCTCGCAGCATTTC

[0113] The multiple nested PCR detection method of Neospora, Brucella and bovine infectious rhinotracheitis virus in the present embodiment comprises the steps:

[0114] 1) extract the DNA of the sample after mixing the brain, liver and lung tissue of the fetal bovine;

[0115] 2) Use the DNA obtained in step 1) as a template, add the outer primer set, and perform the first round of PCR amplification; the system for the first round of PCR amplification is 25 μl, including: 2.5 μl of 10×TranStart Taq Buffer (purchased From Beijing Quanshijin Company), 1.5mmol / L of dNTPs, 0.6μmol / L of each primer in the outer primer set, 1.5U of TranStart Taq DNA polymerase, 1μl of template, and water as the balance; the reaction procedure is: 95 ℃ pre-denaturation for 3 minutes; 95℃ for 30s, 53℃ for 1min, 72℃ for 1min, 35 cycles; 72℃ for 10min; get the first round of amplification products;

[0116] 3) Use the first-round amplification product as a template, add the internal primer set, and per