Qiasymphony dsp virus pathogen midi kit

Peripheral venous blood (8mL), in EDTA-containing tubes, from women with singleton euploid pregnancies and samples from non-pregnant women were obtained from the Translation Genetics Team biobank of the Cyprus Institute of Neurology and Genetics. Plasma samples were obtained from pregnancies between 11^th-14^th weeks of gestation, carrying fetuses of either gender. Within 4 hours after venipuncture, blood samples were subjected to centrifugation at 1,600 X g for 10 min at 4°C. The separated plasma portion was obtained and was re-centrifuged at 16,000 X g for 10 min at room temperature. Following this, plasma samples were stored at -80°C until further processing. Circulating cfDNA was extracted from 4 mL plasma using the QIAsymphony SP instrument and the QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions (QIAsymphony DSP Virus/Pathogen Midi Kit Handbook). Affected samples were generated using samples from individuals diagnosed with Wolf-Hirschhorn, and Potocki-Lupski syndrome (17p11.2 duplication syndrome) that were obtained from the Coriell Cell Repositories (Camden, NJ). Affected spiked-in samples were also generated for 1p36 deletion, Smith-Magenis, Miller-Dieker (MDS) 22q11.2 deletion syndrome and NF1 microdeletion syndromes using DNA samples acquired from the Cyprus Institute of Neurology and Genetics biobank (Table 1).

Cloacal swabs were resuspended with 500 μl PBS containing antibiotics and antimycotics (1X Antibiotic-Antimycotic Solution, Sigma-Aldrich, Saint-Louis, MO, USA) (PBS-A). Nucleic acids were isolated from 300 μl of swab suspension with the QIAsymphony® DSP Virus/Pathogen Midi Kit on a QIAsymphony® SP instrument using a custom protocol provided by Qiagen (Hilden, Germany).
IBDV genome was detected by rRT-PCR targeting the VP4 gene (Peters et al., 2005 (link)) modified to a simple setup, using the QuantiTect® Multiplex RT-PCR Kit (Qiagen®, Hilden, Germany). Briefly, each reaction contained 12.5 μl of 2x QuantiTect® Multiplex RT-PCR Master Mix, 250 nM of each primer, 300 nM of a probe targeting very virulent IBDV strains (FAM – 5′-CAACGCCTATGGCGAGATTGAGAACGTGAG-3′- TAMRA),0.25 μl of QuantiTect® Multiplex RT Mix, 5 μl of template RNA and RNase-free water up to 25 μl. rRT-PCRs were run on Rotorgene 6000 (Qiagen®, Hilden, Germany) under the following cycling conditions: 50°C for 20 min and 95°C for 15 min, followed by 40 cycles at 94°C for 45 s and 60°C for 45 s. Each sample was tested in duplicate. rRT-PCR amplification data were analyzed with the Rotorgene Q series software (Qiagen®, Hilden, Germany).

Before DNA extraction, plasma samples were centrifuged at 16,000 g for 10 minutes at 4^oC in order to remove cell debris. Circulating cfDNA was extracted from 2 mL of plasma using the automated QIAsymphony extraction system and a QIAsymphony DSP Virus/Pathogen Midi kit (QIAGEN, Hilden, Germany) or using a QIAamp DNA Circulating Nucleic Acid kit (QIAGEN) according to the manufacturer’s instructions. Circulating cfDNA was eluted into 90 μL of elution buffer and stored at 4 ^oC. Eluted cfDNA was quantified by SYBR Green I real-time PCR of human LINE-1 sequences20 (link). PCR was performed in 20 μL reaction volume, containing 3 μL extracted cfDNA, 0.5 μM each of the forward primer (5′-TCACTCAAAGCCGCTCAACTAC-3′) and the reverse primer (5′-TCTGCCTTCATTTCGTTATGTACC-3′) and 1x iTaq SYBR Green Supermix (Bio-Rad, Hercules, CA). Amplification was carried out as follows: 2 minutes at 94 ^oC and 35 cycles of 10 seconds at 94 ^oC and 15 seconds at 58 ^oC and 15 seconds at 70 ^oC. A standard calibration curve was valid for 4-fold serial dilution of human genomic DNA (Promega, Madison, WI) up to 8 ng/reaction. Each sample was tested in triplicate.