Cador pathogen 96 qiacube ht kit

RNA and DNA were extracted from the nasal swab samples using the extraction robot QIAcube HT (QIAGEN, Hilden, Germany) and the Cador Pathogen 96 QIAcube HT kit (QIAGEN) using the manufacturer's instructions. Before nucleic acid extraction, nasal swab samples were prepared by centrifuging 400 μl of each individual sample or pool for 5 min at 9,000 × g at room temperature (15–25°C), and 200 μl of the supernatant was subsequently used for extraction. Positive and negative (nuclease-free water; Amresco, Cleveland, OH) controls were included in each extraction. The nucleic acids were stored at −80°C until further analysis.
RNA and DNA were extracted from 10% fecal dilutions of the individual samples or from pools consisting of the 10% fecal dilutions using the extraction robot QIAcube HT (QIAGEN) and the Cador Pathogen 96 QIAcube HT kit (QIAGEN). Prior to nucleic acid extraction, one 5-mm steel bead was added to each sample or pool and the samples or pools were homogenized in a TissueLyser II (QIAGEN) for 20 s at 15 Hz. The homogenate was centrifuged for 90 s at 6,700 × g, and 200 μl of the supernatant was used for extraction. Positive and negative (nuclease-free water; Amresco) controls were included in each extraction. The nucleic acid extractions were stored at −80°C until further analysis.

Total DNA was isolated from the interdental brushes using the QIAcube HT Plasticware and Cador Pathogen 96 QIAcube HT Kit (Qiagen, Hilden, Germany), according to the manufacturer’s guidelines. The elution volume used in this study was 150 μL. DNA quality and quantities were measured using an ultraviolet spectrophotometer at 260 and 280 nm. The DNA sample was considered pure if the A260/A280 ratio was in the range of 1.8–2 and the A260/A280 ratio was in the range of 2–2.2.

RT-PCR assays were utilized to monitor viral loads in plasma, cerebrospinal fluid, cervicovaginal swab, colorectal swab, urine and lymph node. RNA was extracted with a QIAcube HT (Qiagen, Germany). Serum samples were extracted using the cador Pathogen 96 QIAcube HT Kit, and tissue samples were lysed in Qiazol, using the Tissuelyser II (Qiagen, Germany), chloroform treated and extracted with the RNeasy 96 QIAcube HT Kit. The wild-type ZIKV BeH815744 Cap gene was utilized as a standard. RNA standards were generated using the AmpliCap-Max T7 High Yield Message Maker Kit (Cell Script) and purified with RNA clean and concentrator kit (Zymo Research, CA, USA). RNA quality and concentration were assessed by the BIDMC Molecular Core Facility. Log dilutions of the RNA standard were reverse transcribed and included with each RT-PCR assay. Viral loads were calculated as RNA copies per milliliter or VP per microgram of total RNA as measured on the NanoDrop (Thermo Scientific, Waltham, MA, USA). Assay sensitivity was >100 copies/ml and > 1 copy/μg total RNA.