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fm to am nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor

by:SNK     2019-09-16
A sensitive method for DNA constant temperature amplification was developed for detecting DNA at fM to aM concentrations for pathogen identificationstick-
Coated metal micro-fluid biological reactor.
Monitoring of microdna amplification using portable co-focused optical detector
Analysis of nano-reactions in real
Time, there is fluorescence collection near the optical diffraction limit. The non-stick-
The surface contact point of the coated metal micro-fluid reactor is 103 °, which is basically inert to the biological reaction
Molecular and DNA amplification can be performed with a minimum reaction volume of 40 nl.
Constant temperature nucleic acid amplification for identification of non-Mycoplasma pneumoniastick-
A coating micro-fluid reactor can be performed with a minimum DNA template concentration of 1.
At three o\'clock A. M. , the limit for detection of three genomic DNA was obtained.
By expanding the target from several copies of a single bacterial genomic DNA, this microfluid reactor provides a promising method for molecular diagnosis of clinically relevant pathogens.
Purchased at Biotron EvaGreen (California, USA).
Bst dna pcr, large fragments and ThermoPol reaction buffer from New England Biolabs Co. , Ltd (Beijing, China).
Buy MgSO from Beijing chemical reagent company (Beijing, China).
Get dNTPs from TaKaRa biotech. , Ltd (Dalian, China).
End marks and marks-
From Fermentas Inc . (
Burlington, Canada).
Synthesis of Invitrogen by nucleotide primers (Shanghai, China).
Purchased from Fluka Sigma-for the milk proteinAldich Inc (Missouri, USA). The non-
Get the rod coating from Shenzhen sovein Technology Development Co. , Ltd. Ltd (SHENZHEN, China).
Other reagents are analytical grade.
The design of oligomeranian primers for constant temperature amplification analysis according to the P1 gene sequence from Ain sequence (Accession No. M18639).
6 primers, including 2 ring primers (LF and LB)
Two foreign primers (F3 and B3)
, And two internal primers (FIP and BIP)
To identify eight different regions on the target sequence ().
BIP consists of a complementary sequence of B1 and a sense sequence of B2, and FIP consists of a complementary sequence of F1 and a sense sequence of f2.
The primer sequence is listed in.
FH is provided by the People\'s Hospital of Peking University (Beijing, China).
Genomic DNA of FH was extracted and purified using the QIAamp DNA mini kit (Qiagen Inc. , CA, USA).
Measurement of DNA concentration using ND-
1000 optical meter (
Nano drop Technology Co. , Ltd. , DE, USA).
The number of genome copies is calculated by molecular size 0. 8u2005Mbp. A 25-
Isothermal constant temperature nucleic acid amplification analysis for molecular diagnosis of pathogens includes 0.
F3 and B3 are 2 μm each, 1.
FIP and BIP are 6 mu m each, 0.
LF and LB of lf dna pcr are 4 μm, 8 u2005 U, large fragment, 0. 1u2005mM dUTP, 0. 4u2005mM dNTPs, 0. 5u2005mg/ml BSA, 0. 6× EvaGreen, 0.
8 u2005 M Betaine 6 u2005 mM MgS0 months. 1u2005U/ml Uracil-
DNA glucose-based enzyme, 10mm (NH)S0, 20u2005mM Tris-HCl (pH 8. 8 at 25°C), 10u2005mM KCl, 0. 1% Triton X-
100, template DNA (2u2005μL).
When lamp, LF and LB are used to facilitate the amplification reaction, protein is used to reduce the number of reactors, acil-
DNA glucose-based enzymes and dUTP are used to eliminate contamination, EvaGreen as the real-
Time fluorescence was reported on the amplified dsDNA, and the amplification efficiency was improved by using 6 mM MgS0. The 25-
Using ABI 7700 Fast Real-for the analysis of μ l constant temperature amplification in Eppendorf tubes
Time PCR System (
ABI, Missouri, USA).
The reaction mixture was incubated at 37 °c for 5 min, then heated to 65 °c and maintained at a constant temperature of 65 °c for 40-45 min, the reaction is then finally heated to 80 °c for 5 minutes to terminate.
All constant temperature amplification analysis on the micro-flow control chip is used with 25-
Epeppendorf tube but scaled to the appropriate volume.
The metal micro-flow control chip is made of aluminum (Al)
CNC machining center using computer.
Compared to ordinary PC, pmma5, silicone rubber, glass and silicon, Al has excellent thermal properties, is easy to process, and has a rich method of surface treatment.
Here we explore a non-
The surface treatment of the adhesive coating makes the surface of the Al micro-flow control chip inert for the sensitive and stable constant temperature DNA amplification in the micro-medium
Nano reaction analysis, which is not
A rod coating is formed with a polymer silica layer (SiO–SiO)
Combined with PTFEPTFE)(CF–CF).
Structure of micro-
As shown, the nanoliter fluid chip is designed with a chip diameter of 60mm and 2. 5–3.
The thickness is 0mm.
The fixed hole in the center of the chip is used to fix the chip on the rotating stage of the detection system.
Both the inlet and outlet holes are 1.
With a diameter of 2mm, reagents and samples are loaded through the Eppendorf head.
Producing cells with different micro-reactors
Nanoliter volume of 40 nl to 7 u2005 μ l, the channel is carved in different sizes (0. 5–0. 1u2005mm)
Reduce reagent consumption in width and depth.
The buffer cell is 3.
Diameter 0mm, 0.
5mm depth for collecting air from the injected sample to eliminate bubbles in the reactor cells.
The inlet hole is connected to the buffer unit through the channel on the back of the chip. Micro-
The nanoliter flow control chip was manufactured through the process shown in.
First of all, use computer numerical control machining center JT-produce reactor unit, channel, buffer unit, inlet hole, outlet hole and fixed hole on aluminum plateM960L (
Jiatai CNC Co. , Ltd.
China Quanzhou Co. , Ltd)
, And primary Micro
Get the Nano flow control chip.
Second, the surface is micro
The nano-fluid CHIP covers non-
The coating is glued to a thickness of about 10-20 μm, making the surface of the chip smooth and not lively to DNA molecules.
Then, clean the chip with waterless ethanol and dry it with nitrogen.
Finally, a thin film of polyester (~0. 1u2005mm)
From ABI company is tightly attached to Micro
Nano-fluid chip for encapsulation. Metal Micro
Nano-flow control chip is coated with non-
Paste the coating using the following non-manufacturing methods
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