Specific primer pair for identifying processed auxiliary material turtle blood, kit and application
A technology of specificity and primer pairs, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., to achieve the effect of long storage period, convenient means and strong subjectivity
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Embodiment 1
[0041] Example 1 Design of specific primer pairs
[0042]Using the CO I universal primer HCO2198 / LCO1490, a band of about 700 bp was amplified in 4 batches of processed soft-shelled turtle blood samples and successfully sequenced. According to the measured CO I sequence (as shown in SEQ ID NO: 1), all registered files were downloaded from NCBI. The Clustal W program was used for multiple sequence alignment of the CO I sequences of the turtle family and common animals, such as donkey, cattle, chicken, sheep, pig, and rabbit, to analyze the differential SNP sites. Three specific SNP sites were obtained from it, among which 306 is C (with GenBank sequence JF700184.1 as reference), 394 is G, and 520 is T, which are unique SNP sites of Chinese soft-shelled turtle, which can be used for processing soft-shelled turtle blood 4 pairs of Chinese soft-shelled turtle-specific identification primers were designed by Primer Premier 6, and based on ZHB1, ZHB2 was designed by introducing a mi...
Embodiment 2
[0052] Example 2 Optimization of PCR amplification conditions
[0053] The specific primer set obtained according to Example 1 and the T of the amplified product m The PCR reaction system and amplification program were set according to the value and length, and the PCR amplification was carried out with the genomic DNA of the processed turtle blood and the commonly used animal blood samples as the template. details as follows:
[0054] The PCR reaction system was 10 μL, genomic DNA extract was 100 ng; 2 × Rapid Taq Master Mix 5 μL; primer pairs (10 μM) for each of the forward and reverse sequences were 0.2 μL; double distilled water was added to 10 μL.
[0055] The PCR amplification procedure was pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 sec, annealing at 62°C for 15 sec, extension at 72°C for 15 sec, 29 amplification cycles, and thorough extension at 72°C for 3 min.
[0056] a. The PCR amplification procedures of the aforementioned set of PCR identific...
Embodiment 3
[0068] Example 3 Identification by PCR amplification test of soft-shelled turtle blood excipients and specific DNA sequences in whole blood of animals of different origins
[0069] (1)Material
[0070] a. Collect 12 batches of Chinese soft-shelled turtle blood samples in the Chinese soft-shelled turtle breeding base, each sample is about 30 mL; collect donkey blood, cow blood, chicken blood, sheep blood, pig blood, and rabbit blood samples in Xinyi and Nanjing, Jiangsu, each sample The sample was about 5 mL (see Table 1).
[0071] b. Reagents: agarose (Bio Froxx); Trans DNA Marker II (Beijing Quanzhijin Bio); 2× RapidTaq Master Mix (Nanjing Novi Biotechnology Co., Ltd.); FastPure Blood DNA Isolation Mini Kit V2 (Nanjing Novi Biotechnology Co., Ltd.) Zan Biotechnology Co., Ltd.); Ultra GelRed (1000×) (Nanjing Novizan Biotechnology Co., Ltd.); Tris-acetic acid electrophoresis buffer (50×TAE) (LEAGENE); primers are Shanghai Sangon Biotechnology Co., Ltd. synthesis.
[0072] c....
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