A primer set, method for detecting blood sample species and application
A blood sample and primer set technology, which is applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve the problems of cumbersome operation, low extraction efficiency and low detection sensitivity of molecular biology methods, and achieve Improved detection specificity, less DNA loss, and simple operation
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[0052] Preparation of indicator for loop-mediated isothermal amplification technology: configure fisetin at a concentration of 1.25-2.5 mM, flavone at a concentration of 2.5-3.75 mM or berberine hydrochloride at a concentration of 1.25-3.75 mM. Add 1 μl of the above indicator per 25 μl reaction system. That is, the final actual dosage: the addition amount of berberine hydrochloride is 50-150 μM, the final concentration of flavone is 100-150 μM, and the addition amount of fisetin is 5-100 μM. The solvent of the indicator of the loop-mediated isothermal amplification technology is DMSO. DMSO has the functions of increasing the solubility of the indicator, reducing the background noise, reducing the Tm value of the nucleic acid, making it more volatile and improving the specificity.
[0053] Treatment of primers: The human-specific primers synthesized by Jiangsu Jinweizhi Biotechnology Co., Ltd. were prepared with deionized water to prepare 25 with FIP and BIP concentrations of 2...
Embodiment 1
[0063] Embodiment 1: Following the above technical solution, this embodiment provides specific screening methods and screening results of primer sets.
[0064] According to the query of the NCBI database, the conserved sequence of the human-specific gene Homo sapiens notch 2 N-terminallike A (NOTCH2NLA) is: GenBank: BC019835.1 (https: / / www.ncbi.nlm.nih.gov / nucleotide / BC019835. 1?report=genbank&log$=nuclalign&blast_rank=3&RID=2DR4CPJ6014), the DNA extracted from human oral swabs was used as a template, and primers were designed on the website http: / / primerexplorer.jp / e / , and the sequences of 5 groups of primers were shown in Table 1. The primer synthesis was completed by Suzhou Jinweizhi Biotechnology Co., Ltd. The amplification efficiency and specificity were compared. Finally, a set of primers with the best specificity and amplification efficiency was obtained.
[0065] Table 1 Primer numbers and primer sequences of each group
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[0068] The specifici...
Embodiment 2
[0069] Embodiment 2: This embodiment provides a method for detecting blood sample species, including the following steps:
[0070] Step 1: Add a DNA rapid extraction reagent to the blood stain sample, and then incubate in a metal bath to obtain a DNA template;
[0071] DNA rapid extraction reagent includes lysate and lysate, the lysate includes 500FU / mL nattokinase, 10U / mL plasmin, 30μg / mL adenosine concentration and 1mM EDTA, the lysate includes 10mg / mL Proteinase K, 1% Triton X-100 and 5 mM CaCl 2 ; wherein "%" refers to the mass percentage by volume. Specific operation steps: Weigh 250 mg of nattokinase, 10 mg of plasmin and add 8 mL of deionized water, then add 100 μL of 300 μg / mL adenosine and 50 μL of 0.5M EDTA, and add ionized water to make up the volume to 10 mL to obtain a solution. Measure 100 mg Proteinase K, 100 μL Triton X-100, 500 μL 0.1 M CaCl 2 solution, and dilute to 10 mL with deionized water to obtain a lysis solution.
[0072] The bloodstain is formed b...
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