1 Reagents
Inclusion buffer: 20 mmol/L Tris-HCI (pH 9.5) containing 150 mmol/L NaCl and 0.02% NaN3;
Washing solution: 20 mmol/L Tris-HCI (pH 7.5) containing 150 mmol/L NaCl 0.1 mmol/L EDTA and 0.1% Tween20;
Dilution solution: 20 mmol/L Tris-HCI (pH 7.5) containing 150 mmol/L NaCl0.45% skimmed milk and denatured salmon sperm DNA (0.1 mg/ml)
2 Operation
1) Encapsulation
Dilution of the encapsulation buffer with the antigen (BSA), add to microtitre plate (45μl wells), overnight at 4°C (about 15h), and wash the plate with washing solution 3 times×5min.
2) Confinement:
Add 200μl per well of 20mmol/L Tris- containing 4.5% skimmed milk and denatured salmon sperm DNA (1mg/ml), 0.1mmol/L EDTA Hcl (pH 7.5) (containing 150 mmol/L NaCl buffer, incubated at 37°C for 80 min, and washed the plate several times.
3) Antigen-antibody reaction:
Dilute monoclonal anti-BSA antibody with release solution 1:8000, add 50 μl to each well, and wash the plate wells 15 times×10min at room temperature (~22℃) for 45min to remove unbound antibody molecules.
4) Chain-affinity protein A binding reaction:
Add 50 μl of chain-affinity-protein A chimera that has been bound to biotin-pUC19 diluted with diluent solution per well for 50 min at room temperature (～22°C), so that chimera-pUC19 binds to the antigen-antibody complexes in the solid phase, and then wash the plate 15 times × 10 min, then wash the plate with NaN3-free Then the plate was washed 15 times×10min, and then washed 3 times with NaN3-free TBS, and then the microtitre plate could be used for the PCR reaction later.
5) PCR
Experimental conditions 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 8.3 at 20°C), 1.5 mmol/L MgCl2, gelatin (10 μg/ml), 0.8 mmol/L dNTPs (0.2 mM for each), 2 μM primers (1 μM for each primer) and TaqDNA polymerase (50 U/ml).
PCR cycle Prior to PCR, the above reaction mixture was irradiated with ultraviolet (UV) light (254 nm), and then added to microtitre plate wells at 40 μl per well, covered with 20 μl of UV-irradiated paraffin wax, and subjected to a PCR cycle on a PCR instrument at the following temperatures: starting denaturation at 94°C for 5 min; 30 cycles: denaturation at 94°C for 5 min, annealing at 58°C 1min, extension 72℃ for 1min, and final extension 72℃ for 5min, the PCR product obtained was a specific 261bp fragment. ⒈ Dilute the bacterial solution to a certain concentration with 0.85% NaCl.
Peake Add 50μl to the microtiter plate at 4℃ overnight. Also set up a negative control (add 50 μl 0.85% NaCl in another well).
3) with 400 μl of 0.05% Temperature 20pBS (hereinafter referred to as TPBS), washed five times.
Sung with 2.25% 100μl of normal goat serum (NGS) PBS for 2 hours.
Carefully wash three times with TPBS containing 0.15% NGS.
Select 100 μl of monoclonal antibody (containing 100 μg of fresh fish sperm DNA) appropriately diluted with 0.75% NGS and incubate for 30 min at room temperature.
Borrow Wash five times with TPBS.
Add 50 μl of moderately diluted biotinylated anti-mouse IgG antibody and incubate for 30 min at room temperature.
Wash five times with TPBS.
Sneakers Add 60 μl of appropriately diluted biotinylated DNA and affinity protein, and incubate for 30 min at room temperature.
Wash five times with TPBS, and then wash three times with HPLC-grade water.
Application form for PCR amplification: add 50 μl of PCR reaction solution (containing T3 and T7 primers), 95 ℃ for 60sec, 50 ℃ for 110sec, 72 ℃ for 110sec, and cycle 30 times. Take 10μl of PCR product on 1.7% agarose gel for electrophoresis, and compare with the molecular weight standard of nucleic acid, the electrophoretic band at the corresponding position is positive. The electrophoresis results were photographed for quantitative analysis when necessary. 1) Gel detection system Quantification of ethidium bromide-stained agarose or polyacrylamide gel by gel scanner or computer-assisted video equipment, quantification of radiolabeled amplification products by radioautography, and recent detection of fluorescently labeled nucleic acids by automated DNA sequencer, which can accurately measure the size of the amplification products, and the sensitivity of its detection is up to the level of fg, but due to the automated DNA sequencer and fluorescently labeled primers, it can be possible to measure the size of the products accurately, and its detection sensitivity can reach fg level. DNA sequencer and fluorescently labeled primers are extremely expensive, so they are now used only for research.
2) HPLC detection system The advantages of this method are that the PCR product does not need to be purified, and the sensitivity can be up to the fg level for unlabeled products, and the detection limit for labeled products may be even lower. hplc is able to differentiate between the sizes of different molecules, which may allow us to use different sizes of internal standards to measure the efficiency of amplification.
3) Solid phase assay system The most commonly used is the 96-well polystyrene microtitre plate, which can be used for instrumental colorimetry or reading, and can be used to encapsulate the titre plate by hydrophobic interactions with the amphiphilic molecules, and this solid-phase system can specifically bind to biotin or biotinylated molecules and can be used to quantify biotinylated PCR products, such as double-labeled amplified products with biotin and digoxin, which can be quantified with the microtitre plate method. There are three ways to label PCR products with both biotin and digoxin: (i) by adding both digoxin and biotin-labeled deoxyribonucleotide analogs (DIG-/Bio-dUTP) to the reaction mixture; (ii) by adding biotinylated primer 1 and DIG-dUTP; and (iii) by adding biotin primer 1 and digoxin-labeled primer 2. Quantification of the doubly-labeled PCR products can be achieved as follows precipitated with ethanol to remove unbound markers, and then added to the affinityin-coated microtitre plate warmed for at least 2 hours, washed several times, and then warmed with the DIG antibody:AP conjugate, which produces different colors depending on its substrate. affinityin-mediated solid-phase capture of biotin-labeled target molecules is an effective, flexible, and easy-to-operate technique that will be a key technology for quantitative PCR .
4) SPA (Scintillation Proximity Assay) system The fluorine microspheres encapsulated with affinity elements are used as a solid-phase carrier, and biotin-labeled primers are used to mix fluorinated nucleotides during amplification, and after the completion of the amplification, the encapsulated fluorine microspheres are added to capture the biotinylated PCR products, and the capture process can be made to be closely bound to the fluorine microspheres, and the fluorine is excited by deuterium to produce light pulses, which can be used to scintillation. The fluorine is excited by deuterium to produce light pulses, which can be measured by a scintillation counter. This method has a greater linear range than the colorimetric method.
5) Dot hybridization detection system The amplification product is fixed on the surface of the nylon membrane after denaturation, and hybridizes with the labeled DNA probe, and the sequence-specific oligonucleotide probe can also be fixed on the membrane through the poly T (poly T) tail, and hybridizes with the denatured amplification product that has been labeled, and the kinetics of the reaction is close to that of the liquid phase, because the target gene is not directly bound to the surface of the membrane. The reaction is fast, usually using a biotinylated probe or amplification product, with an affinity protein-AP conjugate that produces a color spot, which is quantified by comparing the color intensity with a standard of known concentration.
6) Electrochemiluminescence detection system The amplification product is conjugated to magnetic beads by the affinity-biotin system, then hybridized to a 2,2'-bipyridine-ruthenium chelate (TBR)-labeled probe, added to tripropylamine (TPA) solution, and transferred to the detection cell of the electrochemiluminescence instrument, where both TPA and TBR are oxidized instantaneously when the voltage at the electrode is increased to a certain level. When the voltage of the electrode is increased to a certain level, both TPA and TBR are oxidized instantly, and the oxidation of TPA generates an unstable and highly reductive intermediate substance, which can react with the oxidized TBR and make it enter into the excited state, and emit 620 nm light when it changes from the excited state to the ground state, and the intensity of luminescence is directly proportional to the amount of the TBR-labeled material, and the PCR products are quantified by the luminescence intensity, and the sensitivity of the method is at the level of amol, and the measurement can be automated. The sensitivity of this method is at amol level and can be automated.
7) DNA enzyme immunoassay system Quantification is carried out by combining anti-DNA monoclonal antibody with the amplification product, and then through the enzyme-secondary antibody conjugate. This method does not require labeling of the primers and amplification products, but its widespread use is limited by the susceptibility to cross-reactivity and the high cost of monoclonal antibodies.
8) Laser-induced fluorescence assay The N-hydroxysuccinimide derivative of the fluorescent marker FAM (trade name) is first coupled to the 5'-nucleotide of the forward primer with the aid of an amide linker arm, and then amplified by PCR, followed by capillary electrophoresis of the amplified product, and then quantitatively measured by argon laser photodetection of the fluorescence intensity. The advantages of this method are low sample volume, automated detection, rapidity, sensitivity and high resolution.
In conclusion, the target genes can be quantified by the above methods, and their accuracy can be improved by multiplexing the tube assay and standardizing the data using internal standards. The technique of affinity-mediated solid-phase capture of biotin-labeled target molecules has good prospects for clinical application because of its effectiveness, flexibility, and ease of operation. Quantitative PCR is still technically problematic and is mostly used in research, but there will be an increasing demand for this technique in the diagnosis and treatment of tumors, bacterial, fungal, and viral diseases.