Hepatitis B virus (HBV) detection by PCR is mainly used for the diagnosis of viral hepatitis, efficacy observation and prevention research work.

Operation method

Hepatitis B Virus (HBV) Detection by PCR

Principle

Polymerase chain reaction (PCR) is a new technology that simulates the natural DNA replication process to amplify specific DNA (or RNA) fragments in vitro. In this experiment, the double-stranded DNA to be examined is denatured into single-stranded by 94℃ as template, and then a pair of synthetic oligonucleotide primers are added, which are complementary to the ends of the DNA fragments to be amplified, and then annealed at a low temperature of 55℃, the primers are combined with the template in a complementary manner. Under the condition of 72℃, the primers bound to the template are catalyzed by DNA polymerase, and the four kinds of dNTP in the reaction system are used as raw materials to extend and synthesize two new DNA strands according to the way of base complementary pairing. The amplified DNA can be used as a template for the next round of amplification reaction. Repeating the above cyclic process, after 20-30 cycles, the specific target DNA can be amplified more than a million times. Specific amplification bands can be observed after agarose gel electrophoresis of the PCR product.

Materials and Instruments

Serum Samples
HBV-PCR reaction solution HBV-DNA lysate Positive template Ethidium bromide
PCR Amplifier Electrophoresis Analyzer UV Analyzer

Move

I. Specimen processing

Take 20 μl of mixed serum and 20 μl of lysate, stir well, then 100 ℃ boiling water bath for 10 minutes, and finally centrifuged at 15000 rpm/min for 3 minutes, take 4 μl of supernatant to be examined.

Sample addition and PCR

Take a tube of reaction solution (slightly centrifuged before use), add 4μl of supernatant to be examined or positive control in the bottom reaction solution, mix well and centrifuged at high speed for a moment, then set the PCR instrument, set the program 94 ℃ pre-denaturation for 2 minutes, and then according to the 94 ℃ / 30 seconds, 55 ℃ / 30 seconds, 72 ℃ / 60 seconds amplification of 35 cycles.

Electrophoresis and judgment of results

Take 15μl of the reaction solution, after 2% agarose gel electrophoresis (5V/cm) for 30 minutes, observe the results under the UV lamp, if there is an orange-yellow band at 410bp, then HBV is positive.

Caveat

1. After adding all the reagents in the reaction tube, the amplification should be carried out immediately to avoid the formation of excessive dimers.

2. Positive template can be replaced by positive serum, the treatment method is the same as the above.

3. The positive template and DNA extraction solution should be fully melted and centrifuged before use.

4. the surface of the reaction is also a solid capping agent, electrophoresis sampling from the bottom of the reaction tube to wash the liquid.

5. EB is a strong carcinogen, the operation process should pay attention to protection.

6. Pay attention to aseptic operation to avoid false positives.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/