To begin, thaw all reagents provided in the real-time PCR kit at room temperature. Once thawed, pulse vortex the components for 10 seconds at a moderate speed to mix them. Then, centrifuge at 8, 000G for 10 seconds at room temperature.
Keep the thawed components on a cooling block. Next, prepare a PCR mix for an HBV positive sample, five HBV standards, and no template control. Thoroughly mix the added reagents by gently pipetting seven to 10 times, followed by pulse vortexing, and centrifuge at 8, 000G for 10 seconds at room temperature.
Then, pipette 15 microliters of the PCR mix into each PCR tube. Add 15 microliters of extracted DNA sample, five HBV standards, and nuclease free water as a no template control. Close the PCR tubes and briefly spin in the centrifuge.
Load the PCR tubes in a real-time PCR machine. Now, initiate the thermal cycler software. Choose fluorescent dyes and program the thermal cycler for real-time PCR.
Post run, analyze the amplification plot using a linear scale. Set the PCR reaction threshold within the exponential phase. Maintain consistency in threshold settings for accuracy and reproducibility.
Use the same threshold for all samples on a specific PCR machine. Consider samples with amplification before CD value 38 as HBV positive based on the assay's cutoff. Use five quantification standards for HBV-specific DNA, calibrated against the Fourth WHO international standard for HBV DNA.
Generate a standard curve with these standards to quantify HBV DNA in unknown samples. Interpret values for unknown samples only if the standard curve slope is between minus 3.1 and minus 3.6, the R square value is between 0.99 and 1.0, PCR efficiency is within 90%to 110%and there is no amplification in the negative control.
