Primer combination, probe combination, application of primer combination or probe combination to detection of porcine viruses, detection reagent, kit and detection method
A primer combination and detection reagent technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve problems such as difficult clinical sample detection, improve detection efficiency, reduce workload, The effect of reducing the amount of reagents
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Embodiment 1
[0068] Embodiment 1: This embodiment provides a method for joint detection of five porcine viruses. Specifically, a five-fold fluorescent quantitative PCR detection method was established to establish a joint detection method for five porcine viruses.
[0069] 1. Design of primers and TaqMan probes
[0070] The B646L gene sequences of different African swine fever virus (ASFV) strains published by GenBank (accession numbers: MN393476.1, MN715134.1, MN172368.1, LR743116.1, MK333180.1, MH910495.1, MN336500.2); 3D gene sequences of foot-and-mouth disease virus (FMDV) strains (accession numbers: FJ175661.1, GU384683.1, DQ989323.1, DQ989308.1, FJ175662.1, FJ175665.1, MF372126.1, DQ989320. 1); Nsp2 gene sequence of porcine reproductive and respiratory syndrome virus (PRRSV) strain (accession numbers: NC_043487.1, EF635006.1, EF112445.1, EF641008.1, AY150564.1, MH500776.1, MN119307.1 , KP771777.1); 5´UTR gene sequence of classical swine fever virus (CSFV) strain (accession numbers:...
Embodiment 2
[0128] Example 2: Sensitivity Verification of Five-fold Fluorescent Quantitative PCR Detection Method
[0129] ASFV (5.8 × 10 4 copies / μL), FMDV (8.1×10 4 copies / μL), CSFV (5.1×10 4 copies / μL), HP-PRRSV (9.7×10 4 copies / μL), PRV wild virus (7.6×10 4 copies / μL) Take 10 μL of each nucleic acid and mix evenly (concentrations: 1.16×10 4 copies / μL, 1.62×10 4 copies / μL, 1.02×10 4 copies / μL, 1.94×10 4 copies / μL, 1.52×10 4 copies / μL) after serial dilution of 10 times, 100 times, 1000 times and 10000 times, then used as a template for five-fold fluorescent quantitative PCR detection, each gradient was repeated 3 times, and the minimum detection limit and linearity of the five-fold fluorescent quantitative PCR method were calculated. relation. Amplification results such as Image 6 As shown in Table 11, ASFV is the standard curve of the Texas Red channel (No. 1-Texas Red) regression equation is Y= -3.251X+40.547, and the correlation coefficient (R 2 ) was 0.990, the a...
Embodiment 3
[0132] Embodiment 3: Five-fold fluorescent quantitative PCR detection method specific verification
[0133] FMDV (A type, O type, Asian type I), PRRSV (LV strain, JXA1 strain, VR2332 strain), CSFV (C strain), PRV standard strain, PRV vaccine strain (Bartha -K61 strain), TGEV-PEDV - PoRV was extracted according to step 3 of Example 1, and then the nucleic acid of the ASFV positive sample was used as a template for five-fold fluorescent quantitative PCR detection.
[0134] Test results such as Figure 7 As shown, except ASFV (No. 1), FMDV-A (No. 2), FMDV-O (No. 3), FMDV-Asia Ⅰ (No. 4), PRRSV-JXA1 (No. 5), CSFV (No. 6), PRV Except for wild virus (No. 7), other viruses (No. 8-10) were not detected, indicating that this method has no cross-reaction with other viruses, and can specifically detect ASFV, FMDV, HP-PRRSV, CSFV and PRV wild poison.
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