Primer group and method for meat detection based on seven PCR (Polymerase Chain Reaction) technology
A primer set and meat technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as limiting the accuracy of RT-PCR analysis, and achieve high sensitivity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
specific Embodiment 1
[0068] 1. Sample processing and DNA extraction
[0069] Fresh pure meat from camels, pigeons, chickens, ducks, horses, cattle, and pigs is purchased from local retailers and fresh markets, and immediately shipped to the laboratory on ice for processing. All samples were stored at -80°C to inhibit DNA degradation. Using the EasyPure® Genomic DNA Kit, isolate genomic DNA from meat samples. The DNA concentration was detected by NanoDrop2000 spectrophotometer.
[0070] 2. Design of species-specific primers
[0071] According to the mitochondrial gene accession numbers of each species published by GenBank, the camel cytochrome c oxidase subunit III sequence (No.MH109991.1) and the pigeon NADH dehydrogenase 6 subunit were retrieved from the National Center for Biotechnology Information (NCBI) database. (No.KP168712.1), chicken D-loop (No.MK163565.1), duck ATPase 6 subunit (No.MK770342.1), horse NADH dehydrogenase subunit 4 (No.MN187574.1), Bovine cytochrome c oxidase subunit II ...
specific Embodiment 2
[0102] Primer specificity analysis
[0103] In order to verify the specificity of the primers, single-species DNA and its primer pair were used for single PCR amplification, which was analyzed by gel electrophoresis. The amplified bands of camel, pigeon, chicken, duck, horse, cow and pig were 128bp, 157bp, 220bp, 272bp, 314bp, 434bp and 502bp, respectively. In order to ensure the quality of genomic DNA of the seven species, three pairs of universal eukaryotic primers derived from 18SrRNA, 16SrRNA and 12SrRNA target genes were selected as positive controls respectively ( figure 1 A). In the present invention, all the meat samples produced 99 bp, 240 bp and 456 bp target PCR fragments ( figure 1 B), indicating that the quality of genomic DNA extracted from meat resources is equivalent, ensuring the same amplification efficiency as PCR. Using the genomic DNA of a single species of meat as a template, a single PCR product could be obtained using a primer mixture of 7 species, w...
specific Embodiment 3
[0104] Primer Sensitivity Analysis
[0105] After the specificity analysis of each set of primers, by optimizing the reaction system and procedures, a seven-fold PCR system was finally established using 7 pairs of species-specific primers. To analyze the dynamic range and limit of detection (LOD) of multiplex PCR reactions, template DNA was diluted in steps from 10 ng to 0.01 ng for all species (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01) , judging the threshold value of seven-fold PCR technology detection. like image 3 As shown in A, when the template DNA content is between 10ng and 0.01ng, five clear bands at the top can be observed, from top to bottom, pigs, cattle, horses, ducks and chickens. In contrast, 0.025 and 0.01 ng template DNA content produced faint electrophoretic bands. Image Lab TM The software draws the electropherogram based on the electrophoresis bands. Clear bands match complete peak shapes, and weak bands correspond to defective peak shapes. l...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More