Polymerase chain reaction (PCR) is a novel technique that mimics the natural DNA replication process and amplifies specific DNA (or RNA) fragments in vitro. In this experiment, the double-stranded DNA to be tested is denatured into a single strand as a template at a high temperature of 94 ° C, and then a pair of artificially synthesized oligonucleotide primers are added, and the primers are respectively complementary to the ends of the DNA fragment to be amplified, and the temperature is lowered at 55 ° C. Annealing, the primers are complementary to the template. Under the condition of 72 °C, the primers bound to the template were catalyzed by DNA polymerase, and four kinds of dNTPs in the reaction system were used as raw materials to synthesize two new DNA strands in a complementary pairing manner. The amplified DNA can be used as a template for the next round of amplification reactions. The above cycle is repeated, and after 20 to 30 cycles, the specific DNA of interest can be amplified by a million times or more. A specific amplified band can be observed by electrophoresis of the PCR product on agarose gel.
The PCR method is simple and sensitive, and can detect as little as 0.1 pg of DNA of interest. It has been widely used in the detection of a variety of pathogenic microorganisms.
Hepatitis B caused by hepatitis B virus (HBV) infection has a high incidence in China, and HBV is closely related to cirrhosis and primary liver cancer. Therefore, the diagnosis of HBV is extremely important. PCR detection of HBV-DNA sensitivity is significantly higher than traditional serological methods, and can show the replication of HBV in vivo, directly reflecting the patient's blood infectivity, and serological results that are ambiguous or inconsistent with clinical manifestations, PCR technology has Help to confirm the diagnosis.
ã€material】 The serum to be tested, 20 tubes of HBV-PCR reaction solution (20 μl/tube), HBV-DNA lysate, positive template, ethidium bromide, PCR amplification instrument, electrophoresis apparatus, ultraviolet analyzer.
ã€method】 1. Specimen processing:
Mix 20 μl of the mixed serum with 20 μl of the lysate, stir well, and then boil in a 100 ° C boiling water bath for 10 minutes, and finally centrifuge at 35,000 rpm for 3 minutes, and take 4 μl of the supernatant for examination.
2. Loading and PCR:
Take a tube of the reaction solution (slightly centrifuge before use), add 4 μl of the supernatant or positive control to the bottom reaction solution, mix and centrifuge at high speed for a while, then pre-denaturation at 94 ° C for 2 minutes, then press 94 ° C / 30 Amplification of 35 cycles in seconds, 55 ° C / 30 seconds, 72 ° C / 60 seconds.
3. Electrophoresis and result judgment:
15 μl of the reaction solution was taken and electrophoresed on a 2% agarose gel (5 V/cm) for 30 minutes, and the result was observed under an ultraviolet lamp. If an orange-yellow band appeared at 410 bp, HBV was positive.
ã€Precautions】 1. After adding all the reagents in the reaction tube, it should be immediately expanded on the machine to avoid excessive formation of dimers.
2. The positive template can be replaced by positive serum, and the treatment method is the same as above.
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