Polymerase chain reaction ELISA
Polymerase chain reaction (PCR) is one of the most sensitive methods for detecting DNA in molecular biology techniques, and enzyme-linked immunosorbent assay (ELISA) is the most sensitive and specific detection method in immunology. Polymerase chain reaction ELISA (PCR-ELISA) combines the essence of the above two methods, and becomes a new method with higher sensitivity, more specificity and time-saving than PCR [7]. PCR-ELISA is mainly divided into two categories according to the different biotin and the new labeled substrate of the earthworm: the first is to label the PCR product with biotin and Diguxin simultaneously; the second is to separately expand the PCR product. The product and nucleic acid hybridization probes are labeled.
There are three main ways for biotin and Digoxin to simultaneously label PCR products: 1 Simultaneous addition of biotin and Digoxin labeled deoxynucleotide analog (DIG-Bio-dUTP) in the PCR reaction mixture; 2 Biotinylation Primer and DIG-dUTP; 3 Add biotinylated primer 1 and Digoon new labeled primer 2. The experimental method is as follows: the labeled PCR product is precipitated with ethanol, or the unbound label is removed by a purification column, and added to the well-coated plate of the avidin for 1 to 2 hours, thoroughly washed, and the anti-Glutinous new antibody is added. Then, the alkaline phosphatase conjugate substrate was added and the reaction was terminated, and the result was judged based on the absorbance (A) value. This kind of avidin-mediated solid phase capture biotin-labeled target molecule detection system has higher sensitivity than PCR detection, and is simple, rapid, and reproducible. It can accurately detect a large number of cells in a short time (<4h). Clinical specimens (serum, exudate or formaldehyde fixed specimens); PCR products can be quantitatively analyzed by appropriately adjusting the number of PCR cycles and related parameters and linearly analyzing the yield at multiple time points. Since the detection system is based on double-labeled nucleic acid fragments, adsorption of non-specific PCR products sometimes occurs, causing a high background.
The second type of PCR-ELISA labels the PCR amplification products and nucleotide probes for biotin and digoxin, respectively. PCR amplification with biotinylated primers, followed by addition of avidin-coated ELISA plate, ice bath for 1 h, washing, adding denaturing agent (50 mmol/L NaOH) for several minutes at room temperature, adding hybridization solution and ground. The labeled probe of Guxin was mixed and washed, and then alkaline phosphatase-labeled anti-Glutinic acid antibody was added. After being washed for 1 hour at room temperature, the developer was added and the color reagent was added to read the A value. The method utilizes a biotinylated primer to obtain a biotin-containing PCR product, so that the product can be firmly bound to the avidin coated on the ELISA plate, so that the specific PCR amplification product is adsorbed on the ELISA plate, and then the ground is used. The new labeled probes are hybridized and amplified, which is highly specific, sensitive and reproducible.
The superiority of the PCR-ELISA method is mainly reflected in the following four aspects. 1 Using the strong affinity of the biotin-avidin system, its affinity constant K is about 104 to 107 times that of the antigen antibody, and it is A protein (SPA) to IgF. 108 times. The biotin and avidin are highly specific and rapid in combination, are not easily interfered by the outside world, the complex is very stable, and the dissociation is rarely occurred, which improves the stability of the experiment and shortens the reaction time. In addition, the avidin does not contain a glycosyl group, and the isoelectric point is pH=6.0. Compared with other antigens or antibody-coated ELISA plates, the non-specific adsorption is significantly reduced and the sensitivity is improved. 2 PCR amplification greatly amplifies the positive signal through the amplification itself and the anti-glutinal new antigen antibody reaction, thereby further improving the sensitivity of the detection. The use of uracil nucleotide transferase in 3PCR-ELISA allows the cross-contamination in PCR technology to be greatly controlled without affecting the detection effect, basically overcoming the false positive problem caused by pollution in PCR hybridization technology. The use of 4 labeled probes further enhances the specificity of detection, comparable to radiolabeled Southern hybridization, and the introduction of probes increases the range of applications, genetic polymorphism analysis, virus scores than PCR itself. Type, cloning and other aspects show its unique advantages. In addition, the application of non-isotopic labeling system avoids the harm caused by the use of isotopes, and the whole experimental period is significantly shorter than that of Southern hybridization (about 6h), and the operation is simple and can be used for simultaneous detection of large-scale specimens. Of course, the establishment of PCR-ELISA method has certain requirements, and the hybridization conditions of the PCR product and the probe (the dilution of the PCR product, the hybridization temperature and time) are closely related to the detection sensitivity and specificity.
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