Multiple nested PCR (polymerase chain reaction) detection primer, kit and method for Neospora caninum, Brucella abortus and infectious bovine rhinotracheitis virus
A technology of rhinotracheitis virus and Brucella, applied in the field of molecular genetics, can solve the problems that the existence of antibodies cannot fully reflect the pathogen, the specificity and sensitivity of identification need to be improved, time-consuming and labor-intensive, and achieve a simple and reasonable system composition, Reaction program optimization, the effect of short time consumption
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[0080] Example 1
[0081] The multiple nested PCR detection primers for Neospora, Brucella and Bovine Infectious Rhinotracheitis Virus in this example 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] Inner 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'.
Example Embodiment
[0095] Example 2
[0096] The multiple nested PCR detection kit for Neospora, Brucella and bovine infectious rhinotracheitis virus in this example includes: 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'-TCGTCTCGCAGCATTTCGTC-...
Example Embodiment
[0112] Example 3
[0113] The multiple nested PCR detection method of Neospora, Brucella and bovine infectious rhinotracheitis virus in this embodiment includes the following steps:
[0114] 1) Extract the DNA of the sample after mixing the brain, liver and lung tissues of fetal cattle;
[0115] 2) Using the DNA obtained in step 1) as a template, add the outer primer set to perform the first round of PCR amplification; the first round of PCR amplification system is 25μl, including: 2.5μl of 10×TranStart Taq Buffer (purchased (From Beijing Quanshijin Company), 1.5mmol / L dNTPs, each primer in the outer primer set is 0.6μmol / L, 1.5U TranStart Taq DNA polymerase, template 1μl, the balance is water; the reaction program is: 95 ℃ pre-denaturation for 3 min; 95 ℃ 30 s, 53 ℃ 1 min, 72 ℃ 1 min, 35 cycles; 72 ℃ extension 10 min; get the first round of amplification product;
[0116] 3) Using the first round of amplification product as a template, adding the inner primer set to perform the seco...
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