Nucleic acid, method and kit for multi-liquid phase gene chip for simultaneously detecting and identifying components of three major categories of poultry, fish and ruminants
A liquid-phase gene chip and synchronous detection technology, applied in the field of biotechnology safety detection, can solve the problems of increased detection cost, prolonged detection period, increased manpower, etc. Effect
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Embodiment 1
[0051] Design and preparation of embodiment 1 primers and probes
[0052] Firstly, the specific target genes of poultry, fish and ruminants were screened, and the mitochondrial sequences of poultry, fish and ruminants were selected as target genes for PCR amplification through sequence retrieval and comparison. Download multiple mitochondrial genome sequences of representative species of poultry, fish and ruminants from GenBank, perform comparison analysis, select the conserved region sequences with good species specificity, and then compare the sequences between species. Finally, the 12s rRNA in the mitochondrial genome of birds, fish and ruminants was selected as the target gene for PCR amplification.
[0053] PCR amplification primers and hybridization probes in the liquid-phase gene chip reaction system are designed on the basis of the inventor’s many years of experience, with the assistance of Array Designer 4.0 and DNAMAN 7. The principle of primer design is to ensure th...
Embodiment 2 3
[0057] Example 2 Establishment and optimization of triple liquid-phase gene chip detection method
[0058] The detection process includes triple asymmetric PCR amplification and triple suspension microsphere hybridization detection. It is necessary to conduct research and a large number of experimental explorations on the amplification reaction system (the amount of various general primers and various reagents), the amplification reaction conditions (reaction temperature, time, the number of amplification cycles, etc.), as well as the hybridization reaction system and hybridization reaction conditions. Validation to establish optimal reaction conditions.
[0059] 1. In the triple asymmetric PCR amplification, the key factors such as the amount of three sets of primers, the annealing temperature and time of the PCR reaction, and the number of amplification cycles are mainly optimized. Through a large number of comparisons of the primer concentration gradient, reaction temperatu...
Embodiment 3 3
[0084] The specificity test of embodiment 3 triple liquid phase gene chip method
[0085] The genomic nucleic acid of 41 kinds of animal and plant samples was detected by the triple liquid-phase gene chip method provided in Example 2 of the present invention, including 7 kinds of ruminant and beef powder standards, 12 kinds of fish samples, 8 kinds of poultry samples and chicken Meal standards, samples of 11 other common edible and forage terrestrial and aquatic animal species, and plant protein powders (mainly soybean, wheat, and pea) were tested. The test results are detailed in Table 3 below.
[0086] Table 3 Triple liquid phase gene chip specific test results
[0087]
[0088]
[0089] The test results are consistent with the actual situation of species samples: 7 kinds of ruminants and beef powder standard products are all positive for ruminants, 12 kinds of fish samples are all positive for fish tests, and 8 kinds of poultry samples and chicken powder standard pro...
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