Dual real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting and identifying wild strain and vaccine strain of CSFV (classical swine fever virus) in swine umbilical cord blood and application of dual real-time fluorescence RT-PCR kit
A technology of RT-PCR and swine fever virus, applied in the field of dual real-time fluorescent PCR kits, can solve the problems of inability to effectively distinguish wild strain infection and vaccination, less kits, and long operation time, and achieve direct and effective diagnosis , reliable technical support, and highly specific effects
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Embodiment 1
[0071]Example 1 The composition of the dual real-time fluorescent RT-PCR kit for detection and identification of swine fever virus wild strain and vaccine strain in pig umbilical cord blood
[0072] (1) 2× fluorescent RT-PCR reaction solution (containing enzyme): the raw material was purchased from Nanjing VAZYME Company; the reaction solution contains UNG enzyme system, which can effectively solve the phenomenon of amplification pollution during the amplification process Pollution ability, etc.;
[0073] (2) RT-PCR primer sets CSFV-F and CSFV-R: synthesized by Shanghai Sangon Bioengineering Co., Ltd., prepared with DEPC water to a concentration of 10 μM.
[0074] Upstream amplification primer CSFV-F: 5'-GCCATGCCCATAGTAGGA-3', which is the sequence of SEQ ID NO:1;
[0075] Downstream amplification primer CSFV-R: 5'-CTACTGACGACTGYCCTGTA-3', which is the sequence of SEQ ID NO:2, wherein Y=C / T;
[0076] (3) Specific fluorescent probes CSFV-LV and CSFV-Wt: synthesized by BGI Cor...
Embodiment 2
[0089] Example 2 The method of using the dual real-time fluorescent RT-PCR kit for detecting and distinguishing the wild strain of classical swine fever virus and the vaccine strain in pig umbilical cord blood
[0090] The method for using the kit of the present invention specifically includes the following steps: (1) sample collection; (2) sample processing; (3) RNA extraction; (4) double real-time fluorescent RT-PCR: utilizing the specific primers and probes designed by the present invention Perform double real-time fluorescent RT-PCR detection; (5) judge the result.
[0091] (1) Sample collection
[0092] 1. Take a clean penicillin bottle and cork, wash it, boil and sterilize it for 30 minutes, dry it and collect it for later use;
[0093] 2. When the piglets are born, squeeze the "cord blood" of all the piglets delivered by each sow into a clean penicillin bottle, 3-5 drops per piglet, and squeeze the "cord blood" into the penicillin bottle bottle, sealed;
[0094] Prec...
Embodiment 3
[0119] Example 3 Verification of the dual real-time fluorescent RT-PCR kit for detection and identification of swine fever virus field strains and vaccine strains in pig umbilical cord blood
[0120] 1. Validation of amplification efficiency
[0121] Perform a 10-fold serial dilution of the positive control clone plasmid pEASY-5UTR2 to make its copy number: 10 8 -10 1 copies / μl, each gradient was repeated three times for real-time fluorescent RT-PCR, and a standard curve was made according to the amplification results.
[0122] figure 1 It is the gradient amplification standard curve (FAM channel) of 10-fold dilution of the positive control of the present invention, wherein the parameters of the standard curve are as follows: slope: -3.56, intercept: 39.78, correlation coefficient: 0.998, amplification efficiency: 0.91.
[0123] figure 2 In order to invent a 10-fold diluted gradient amplification standard curve (VIC channel) for the positive control, the parameters of the...
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