A two-color fluorescent PCR primer, probe and method for rapidly distinguishing canine parvovirus vaccine strains from wild strains
A canine parvovirus, two-color fluorescence technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of time-consuming, complicated operation, limited promotion and application, etc., to reduce the time , Good repeatability, high-throughput detection speed
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Embodiment 1
[0088] Example 1 Primers and probes
[0089] After screening a large number of designed primers and probes, it was found that the primer pairs F1 / R1, F2 / R2, and probes P1 and P2 have the best effect on the two-color fluorescence method to distinguish canine parvovirus vaccine strains from wild virus strains. The base sequence is shown below.
[0090] F1: 5'-TATAGCACATCAAGATACAGGAAGA-3' (SEQ ID NO: 1),
[0091] R1: 5'-TCCAATTGGATCTGTTGG-3' (SEQ ID NO: 2),
[0092] Probe P1: 5'-FAM- CCTATCATCTTCCTGTAACAAATGATAG-BHQ1-3' (SEQ ID NO: 3),
[0093] F2: 5'-TGGAAATCACAGCAAACTC-3' (SEQ ID NO: 4),
[0094] R2: 5'-CTAAAGCCATGTTTCCGT-3' (SEQ ID NO: 5),
[0095] Probe P2: 5'-HEX-CGACCGTAAATAATATGGATAAAACTGCGGTCG-BHQ1-3' (SEQ ID NO: 6).
Embodiment 2
[0096] Example 2 Preparation of standard samples, dual-color fluorescence PCR amplification and melting curve analysis
[0097] 1) Extraction of canine parvovirus DNA
[0098] Take samples of disease material infected with CPV. Samples of disease material can be whole blood, feces, rectal swabs and other easy-to-obtain samples that do not cause serious harm to the animal. Whole blood can be directly taken 200μl for use. Stool and rectal test samples are required. Take a certain amount and dissolve it in 1mL PBS hydrochloric acid buffer solution, let it stand for 10-20 min and take 200μl for use; for CPV vaccine, add 3mL PBS hydrochloric acid buffer solution to dissolve, take 200μL for use. Nucleic acid extraction was carried out according to the instructions of MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0 of TAKARA.
[0099] 2) Preparation of standard samples
[0100] In order to verify the feasibility and reliability of the method of the present invention, while constructing a stan...
Embodiment 3
[0132] Example 3 Specific Experiment
[0133] The following is a specific test for the detection method established in the present invention.
[0134] Extract nucleic acid from other common canine diarrhea viruses, such as Canine distemper virμs (CDV), Canine adenovirμs type 2 (CAV-2), Canine coronavirus (CCV), and canine rotavirus Virus (Canine rotavirus, CRV) and Canine parainfluenza virus (Canineparainflμenza virμs, CPIV), reverse transcription of CDV, CCV, CRV and CPIV RNA into cDNA, and use the cDNA of the above virus, the nucleic acid of CAV-2 and the water respectively as The PCR template was analyzed by the PCR amplification reaction and melting curve analysis method in Example 2 above, and compared with the wild canine parvovirus strains CPV-2a, CPV-2b, CPV-2c, vaccine strains CPV-int, CPV- The standard samples of pfu are compared and analyzed, and the melting curve peak pattern is shown as figure 2 Shown.
[0135] From figure 2 It can be seen from A~C that the detection...
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