Fluorescent PCR (Polymerase Chain Reaction) detection kit for African swine fever virus, preparation method and application method
A technology of African swine fever virus and detection kit, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of large error in detection results, unsuitable early diagnosis, and susceptibility to environmental influences, etc., to reduce The effects of missed detection, good repeatability, excellent sensitivity and specificity
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[0072] The embodiment of the present invention also provides a preparation method of the above-mentioned African swine fever virus fluorescent PCR detection kit, including:
[0073] S110. Select the sequence of the highly conserved region in the p72 gene of the ASFV genome, design multiple sets of primers and probes, respectively amplify the multiple sets of primers and probes, and screen the amplification efficiency, probe signal-to-noise ratio, and amplification The curve shape is the best set as the primer and the probe of the ASFV-reaction solution;
[0074] S120, synthesize a recombinant cloning plasmid numbered pUC-p72, the recombinant cloning plasmid containing the target nucleotide sequence of the amplified product of the primer in the ASFV-reaction solution, and using a protein nucleic acid analyzer to analyze the recombinant cloning plasmid The concentration is quantified, and the quantified recombinant cloning plasmid is diluted with TE buffer solution to obtain multiple...
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[0100] Example 1 Preparation method of kit
[0101] Analyze and compare the p72 gene sequence information of 23 ASFV genomes registered in NCBI, select the sequence of the highly conserved region to design three sets of specific primer and probe sequences. For the sequence and label information of each group, please refer to Table 1. See Figure 1 to Figure 3 , Perform amplification experiments on the above three sets of primers and probes to obtain the amplification efficiency, probe signal-to-noise ratio and amplification curve shape of each group. The third group has the best curve shape and sensitivity, and there is no mismatch between the probe sequence and the sequence of some current ASFV strains, so it is selected as the best primer and probe set.
[0102] The cloned plasmid pUC-p72 was synthesized according to the amplified product of the target gene of the ASFV primer obtained by the above screening, and its sequence was GATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATATCAACCCAGTGG...
Example Embodiment
[0129] Example 2 Method of using the kit
[0130] Sample collection: dissecting sick pigs, collecting lung, spleen, liver, lymph nodes and other tissues; or collecting blood and tonsils in vivo.
[0131] Sample pretreatment: Serum and plasma samples do not need to be processed and used directly for testing; other tissue samples need to be processed, take about 1g and grind in a grinding tool, add 1.2mL saline to grind to homogenate, transfer to a 1.5mL centrifuge tube, and centrifuge at 8000g After 5 minutes, take the supernatant and dilute 10 times with normal saline and use it for detection.
[0132] Amplification reagent preparation: Take out the components except the DNA enzyme mixture in the packaging box and place them at room temperature. After they are completely dissolved, shake and mix well for later use; according to the proportion (ASFV-reaction solution 38μL / head + DNA enzyme mixture 2μL / head portion + ASFV-built-in reference 0.5μL / head portion) Take the corresponding a...
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