DNA was extracted from 200 µL diluted whole blood and 200 µL solution extracted from bloodstained materials with a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The extracted DNA was eluted in 50 µL elution buffer and used for genome amplification of the HBV S gene using PCR with AmpliTaq Gold DNA Polymerase (Applied Biosystems LLC, Foster City, CA, USA) in 25 µL aliquots containing 2.5 µL 10 × Gold buffer, 500 M deoxynucleoside triphosphate, 1.5 mM MgCl2, and 0.6 µM primers. The sense primer 5′-GTCTAGACTCGTGGTGGACTTCTCTC-3′ and antisense primers 5′-AAGCCAAACAGTGGGGGAAAGC-3′ were used as previously [10 (link)].
DNA polymerase was initially activated at 95 °C for 11 min for PCR. PCR amplification was performed for 35 cycles at 94 °C for 15 s, 55 °C for 5 s, and 72 °C for 30 s, followed by a final step at 72 °C for 10 min. Amplification was carried out in a PC-320 thermal cycler (ASTEC, Fukuoka, Japan). PCR products were mixed with 6 × loading buffer Orange G and subjected to electrophoresis on a 1.5% agarose gel at 100 V for 30 min. The electrophoresed agarose gel was stained with ethidium bromide (0.5 µg/mL). The image from the agarose gel was captured under UV transillumination on a LAS 4000 mini camera system (Fujifilm, Tokyo, Japan). The limit of detection was 2.6 log copies/mL.
DNA polymerase was initially activated at 95 °C for 11 min for PCR. PCR amplification was performed for 35 cycles at 94 °C for 15 s, 55 °C for 5 s, and 72 °C for 30 s, followed by a final step at 72 °C for 10 min. Amplification was carried out in a PC-320 thermal cycler (ASTEC, Fukuoka, Japan). PCR products were mixed with 6 × loading buffer Orange G and subjected to electrophoresis on a 1.5% agarose gel at 100 V for 30 min. The electrophoresed agarose gel was stained with ethidium bromide (0.5 µg/mL). The image from the agarose gel was captured under UV transillumination on a LAS 4000 mini camera system (Fujifilm, Tokyo, Japan). The limit of detection was 2.6 log copies/mL.