A nucleic acid isothermal amplification reaction system, preparation method and application thereof
A technology of isothermal amplification and reaction system, applied in nucleic acid amplification reaction system, warm amplification reaction system and its preparation field, can solve the problems of inability to realize miniaturization, inability to effectively seal, high manufacturing cost, etc., and achieve high throughput Detection, prevention of solution evaporation, low cost effect
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Embodiment 1
[0055] Preparation of plasmid amplification reaction solution:
[0056] 1× ThermoPol buffer (containing 20.0mM Tris-HCl, 10.0mM KCl, 10.0mM (NH 4 ) 2 SO 4 , 0.1%TritonX-100, pH8.8), 8.0mM MgSO 4 , 0.5mMMnCl 2 , 25.0 μM calcein, 0.4 mM dNTPs, the amounts of each primer in the Pandemic H1N1 (Pandemic H1N1) conserved sequence and FluA conserved sequence are as follows: F3 primer and B3 primer each 0.2 μM, FIP primer and BIP primer each 2.0 μM, LF primer 0.8 μM each of primers and LB primers, 1.0M betaine, 0.32U / μl BstDNA polymerase, a little pUC57 vector inserted with PandemicH1N1 conservative sequence and pUC57 vector inserted with FluA conservative sequence (both of these plasmids were purchased from Shanghai Sangong Bioengineering Technology Services Co., Ltd.) DNA samples were used as templates.
[0057] There are two types of plasmids, which respectively amplify two target sequences related to influenza A virus:
[0058] Epidemic H1N1 conserved sequence:
[0059] GAAA...
Embodiment 2
[0080] The composition of reaction solution, reaction condition, reaction time are all identical with embodiment 1, just pass into the liquid order in glass capillary different, concrete situation of passing into is as follows image 3Shown in (A), and heated with a flexible electric heating plate, reacted at 65°C for 1 hour. Its characteristic is that there are plasmid-containing reaction solutions at both ends of the reaction solution that does not contain plasmids, but they are separated by two water droplets. Under the irradiation of ultraviolet light from an ultraviolet flashlight, the test results that emit purple fluorescence are observed as follows: image 3 In (B) shown.
[0081] Also, using the same plasmid amplification reaction solution in a traditional PCR vial, and performing a LAMP reaction under the same conditions, the same visualization results were obtained.
[0082] This example proves that two sections of water columns are used to separate adjacent reacti...
Embodiment 3
[0084] The composition of reaction solution, reaction condition, reaction time are all identical with embodiment 1, just pass into the liquid order in glass capillary different, concrete situation of passing into is as follows Figure 4 Shown in (A), and heated with a hot plate, reacted at 65°C for 1 hour. It is characterized in that there are two sections of reaction solution on one side of the reaction solution containing the plasmid, neither of which contains the target sequence, one of which does not contain DNA at all, and the other contains DNA of a non-target sequence. Separated by two water droplets, under the irradiation of ultraviolet light from the ultraviolet money detector pen, observe the test results of purple fluorescence as follows: Figure 4 In (B) shown.
[0085] Also, using the same plasmid amplification reaction solution in a traditional PCR vial, and performing a LAMP reaction under the same conditions, the same visualization results were obtained.
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