PCR-ELISA

PCR-ELISA

1. Principle

PCRELISA uses immunological methods to detect PCR products, which is simpler and time-saving than conventional electrophoresis methods and Southern blots. It can process a large number of specimens at the same time and is convenient for automation. The principle of PCR-ELISA is to use biotin (biotin) -labeled primers during PCR amplification, so that the PCR product can be combined with avidin-coated plastic plates, and then probed with digoxin The needle is hybridized with the PCR product, and then it can be detected by an enzyme-linked reaction against digoxin.

2. Materials, methods and results

1. PCR amplification DNA amplification is performed according to the basic PCR technique, except that at least one of the primers used is labeled with biotin at the 5 ′ end. Generally, the biotin-labeled primer at the 5 ′ end can be directly synthesized on the DNA synthesizer.

2. Hybridization of digoxin-labeled probes. 01 μg of digoxin-labeled DNA probe was diluted in 90 μl 50 mmol / L TrisHCl, pH 83, 80 mmol / L KCl. Take 10 μl of PCR product into a tube, heat to 90 ° C, and slowly cool to 67 ° C. Centrifuge for 1 s and keep in a 52 ° C water bath for 1 h.

3 Fix the hybridization product to the plastic plate ①Avidin-coated enzyme-labeled plates available from commercial products can also be coated with avidin according to conventional methods; ②Add 100μl of blocking solution (containing 10mg / ml BSA, 1mg / ml fish sperm DNA in PBS), room temperature for 1h; ③ PBST was washed 3 times; ④ PCR products hybridized with digoxin probe were directly added to the enzyme plate, incubated at room temperature for 1h; .

4ELISA detection ①Dilute anti-digoxin antibody in PBS, add 100μl per well, and incubate at room temperature for 1h; ② Wash 3 times with PBST; ③Dilute the enzyme-labeled secondary antibody in PBS, add 100μl per well, incubate at room temperature for 1h; Wash three times; ⑤ Add 100μl of enzyme substrate to each well, and stop the reaction when the color is suitable; ⑥ Read the result on the microplate reader.

Three, matters needing attention

The sensitivity of this method is comparable to that of radioisotope labels, but the danger of isotopes is avoided. When doing a large number of samples, the reaction solution should be prepared uniformly, and then carefully packed into each reaction tube to avoid contamination and make the conditions of each tube consistent. Non-specific reactions are often caused by direct binding of digoxin-labeled DNA probes to enzyme-labeled plates, so a sufficient amount of fish sperm DNA must be included in the blocking solution.