Qiamp dna mini kit

DNA extraction from ticks were done as described previously [18 ]. In brief, individual adult ticks were cut longitudinally into halves using a clean stainless steel No.10 surgical blade (Swan- Morton, Sheffield, United Kingdom). Halved adult ticks were rinsed with distilled water then dried with Kimwipes paper (Kimberly Clark Worldwide Inc., Roswell, GA) prior to DNA extraction. Halved adult ticks were separately crushed in lysis buffer (buffer ATL) with proteinase K and incubated at 56 °C up to 48 h until the ticks were completely lysed. DNA was extracted using QIAmp DNA Mini Kit (QIAgen, Valencia, CA), according to the manufacturer’s instructions. DNA extraction of human whole blood samples were done by QIAmp DNA Mini Kit (QIAgen, Valencia, CA), according to the manufacturer’s instructions. The DNA of tick and human blood samples were stored at -20 °C until further use.

HBV extracellular DNA and core-associated DNA (core-DNA) were isolated as described previously with minor modifications (Kleines et al., 2003 (link); Gao and Hu, 2007 (link); Cui et al., 2013 (link)). Briefly, the culture supernatant was treated with DNase at 37°C for 30 min, and extracellular DNA was extracted by a QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For isolation of core-DNA, the cells were lysed in lysis buffer (50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% Nonidet P-40) (Gao and Hu, 2007 (link)). After removal of the nuclear pellet by centrifugation, the supernatant was treated with 20 U/ml DNase (Takara, Shiga, Japan), 5 µg/ml RNase (Roche Diagnostics GmbH, Mannheim, Germany), 5 mM MgCl₂ and 5 mM CaCl₂ at 37°C for 3 h to degrade the nucleic acids outside the nucleocapsids. The DNase was then inactivated by the addition of 10 mM EDTA. After the inactivation of DNase, proteinase K (final concentration, 0.6 mg/ml), sodium dodecyl sulfate (SDS; 0.5%) and NaCl (50 mM) were added to disrupt the nucleocapsids. Finally, core-DNA was isolated by a QIAmp DNA Mini Kit (Qiagen). The amounts of extracellular DNA and core-DNA were measured by real-time PCR using Fast SYBR Green Mater Mix (Applied Biosystems) with the HBs-specific primers. The thermal profile was as follows: 40 cycles of 95°C for 1 s and 60°C for 20 s.