Detection of hepatitis B virus by PCR
Polymerase chain reaction (PCR) is a new technology that simulates the process of natural DNA replication 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 Â° C, and then a pair of artificial oligonucleotide primers are added. The primers are complementary to the two ends of the DNA fragment to be amplified. After annealing, the primer and template are complementary. Under the condition of 72 â„ƒ, the primers bound to the template are catalyzed by DNA polymerase, and the four new dNTPs in the reaction system are used as raw materials to extend and synthesize two new DNA strands in the manner of base pairing. The amplified DNA can be used as a template for the next round of amplification reaction. Repeating the above cycle process, after 20 to 30 cycles, the specific target DNA can be amplified more than one million times. After agarose gel electrophoresis of the PCR products, specific amplification bands can be observed.
The PCR method is easy to operate, has high sensitivity, and can detect as little as 0.1 pg of target DNA. It has been widely used in the detection of various pathogenic microorganisms.
Hepatitis B caused by hepatitis B virus (HBV) infection has a high incidence in China, and HBV is closely related to liver cirrhosis and primary liver cancer. Therefore, the diagnosis of HBV is extremely important. PCR detection of HBV-DNA is significantly more sensitive than traditional serological methods, and can show the replication of HBV in vivo, directly reflect the infectivity of patients â€™blood, and have ambiguous or inconsistent serological results. PCR technology has Help to confirm the diagnosis.
[Materials] Serum to be tested, HBV-PCR reaction solution 20 tubes (20 Î¼l / tube), HBV-DNA lysate, positive template, ethidium bromide, PCR amplifier, electrophoresis instrument, ultraviolet analyzer
1. Specimen processing:
Take 20Î¼l of mixed serum and add 20Î¼l of lysate, mix well and boil water bath at 100 â„ƒ for 10 minutes.
Finally, centrifuge at 15000 rpm / min for 3 minutes, and take 4 Î¼l of supernatant for inspection.
2. Sample adding and PCR:
Take a tube of reaction solution (slightly centrifuge before use), add 4Î¼l of supernatant to be tested or positive control to the bottom reaction solution, mix and centrifuge at high speed for a while, then pre-denature at 94 â„ƒ for 2 minutes, then press 94 â„ƒ / 30
35 cycles in seconds, 55 Â° C / 30 seconds, 72 Â° C / 60 seconds.
3. Electrophoresis and result judgment:
Take 15Î¼l of the reaction solution and observe the result under ultraviolet light after electrophoresis on 2% agarose gel (5V / cm) for 30 minutes. If an orange band appears at 410bp, HBV is positive.
1. After adding all reagents to the reaction tube, it should be immediately amplified on the machine to avoid excessive dimer formation.
2. The positive template can be replaced with positive serum, the processing method is the same as above
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