High pure viral nucleic acid extraction kit

MSCs were plated on 6-well plates (1 × 10⁵/well) and were infected with Adtrack at 500 MOI. On the next day, cells were washed twice with PBS and transduced with LentiR.E1A (lentiviral expression vector for E1A was shown in Additional file 1: Figure S2) with 8 μg/mL polybrene (Sigma). Co-infected MSCs and corresponding supernatants were harvested at indicated time, from which the adenoviral DNA was extracted using High Pure Viral Nucleic Acid Extraction Kit (Roche). Digital PCR was performed by QX200 Droplet Digital PCR system to examine the exact copy number of hexon gene (nucleotides 21049–21334 of Ad5), representing the quantity of viral particles. The primers were designed as follows: 5′-GGTGGCCATTACCTTTGACTCTTC-3′ and 5′-CCACCTGTTGGTAGTCCTTGTATTTAGTATCATC-3′. Finally, the DNA copy numbers were converted to total adenovirus particle counts by multiplying a certain dilution ratio.

HBV DNA isolation was performed using High Pure Viral Nucleic Acid Extraction Kit (Roche, USA) according to the manufacturer’s instructions. The isolation procedure was based on spin-column method. The final elution volume of 50 µL containing viral RNA from each sample was stored at -20ºC for long-term usage.

HUMSCs were seeded in 6-well plates at a density of 1×10⁵ cells/well and incubated overnight at 37°C. On the next day, the HUMSCs were infected with Ad-hTERTp-IL24 at multiplicity of infection (MOI) 500 for 6 hours. Then, the culture medium was replaced with fresh medium containing lentiviral supernatants (LentiR or LentiR.E1A) at MOI 8 with 8μg/ml polybrene (Sigma, Santa Clara, CA). Twelve hours later, the medium was replaced. HUMSCs and supernatants were harvested after the indicated periods of lentiviral infection, and used for adenoviral DNA preparation using the High Pure Viral Nucleic Acid Extraction Kit (Roche, Basel, Switzerland). Quantitative real-time PCR was performed using ABI PRISM 7500 real time PCR system. SYBR green technology was used to detect a 286-bp-long amplicon (nucleotides 21049-21334) within the conserved region of the Ad5 hexon gene. The primers were designed as follows: 5′-GGTGGCCATTACCTTTGACTCTTC-3′ and 5′-CCACCTGTTGGTAGTCCTTGTATTTAGTATCATC-3′. PCR cycles were programmed according to the manufacturer's instructions for SYBR Premix Ex Taq reagent (Takara, Dalian, China). The standard curve for adenovirus quantification was generated by serial dilutions of pAdTrack plasmid. The experiments were repeated for three times.

Briefly, DNA was extracted from 200μl of serum sample by using a High Pure Viral Nucleic Acid extraction Kit (Roche, Germany) according to manufacturer’s instructions. Six nested PCRs were performed to obtain the complete genome of the isolates (S1 Table). Samples were sequenced using an ABI3730XL Sequencer (Applied Biosystems, USA). Sequences were edited and aligned with BioEdit v.7.2.0 [16 ]. Sequences were deposited in Genbank with the accession numbers KJ843163 -KJ843218 (S2 Table).

To confirm the presence of other pathogens in the Kagoshima sample, detection of agents causing diarrhea using our real-time PCR system, referred to as “Dembo-PCR,” was performed [46 (link)]. This system can identify 19 species of pathogens, including virus, bacteria and protozoa. Briefly, viral DNA and RNA were extracted by high pure viral nucleic acid extraction kit (Roche Diagnostics GmbH, Mannheim, Germany) and bacteria and protozoa DNA were extracted by QIAamp Fast DNA stool mini kit (QIAGEN, Hilden, Germany). Nucleic acids extracted by each kit were subjected to Dembo-PCR, according to a previous report [46 (link)].