Magmax viral rna isolation kit

Fecal swabs collected with Synthetic-Tipped Applicators (Thermo Fisher Scientific, Waltham, MA) were re-suspended by being swirled in 1 ml PBS (Thermo Fisher Scientific, Waltham, MA), followed by total genetic materials extraction using MagMAX™ viral RNA isolation kit (Life Technologies, Carlsbad, CA) on a Kingfisher magnetic particle processor (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions with minor modification as described previously [2 (link)]. Specifically, extracted DNA/RNA was eluted into 50 μl elution buffer. Extracted nucleic acids were then treated by RNase-free DNase I (Qiagen, Hilden, Germany) for 15 min at ambient temperature to remove DNA, and was then purified by Agencourt® RNAclean® XP Beads (Beckman Coulter Life Sciences, Indianapolis, IN) following the manufacturer’s instruction.

Virus isolates tested positive by SV-A qRT-PCR were processed for Next Generation Sequencing (NGS) for complete genome sequencing based on the method described by Wang et al 2003 [20 ]. Briefly, total nucleic acid was extracted using the MagMax Viral RNA isolation kit (Life Technologies). RNA was quantified using a Qubit flourometer (Invitrogen). First strand synthesis of cDNA was performed with random primers and Superscript III reverse transcriptase (Invitrogen) for 50°C for 30 min, followed by incubation for 5 min at 65°C. Immediately afterwards, an additional 1ul of Superscript III was added to the reaction mix and incubated for 30 min at 50°C. Second strand synthesis was performed by addition of Sequenase enzyme (Affymetrix Santa Clara, CA); followed by a ramped incubation to 37°C for 16 min followed by 2 min at 94°C. Subsequently, the 37°C incubation was repeated with the addition of Sequenase, and the final product was amplified using TaqR mastermix (Clontech/Takara) using manufacturers’ recommended conditions. The resulting double stranded amplicon was processed for producing the sequencing library using a Nextera DNA sample preparation kit (Illumina) according to the manufacturers’ protocol. Sequencing was performed with a 600-cycle MiSeq sequencing kit (v3), and run on a MiSeq (v2) instrument.

Viral RNA was extracted from 100 μL of serum and 1 g of lung samples using a viral RNA extraction kit (MagMAX™ Viral RNA Isolation Kit, Life Technologies) and a total RNA extraction kit (Hybrid-RTM, GeneAll, Seoul, Korea) according to the manufacturer’s instructions. The viral loads in sera and lungs were measured by performing real-time RT-PCR employing a one-step reverse transcriptase kit (AgPath-IDTM One-Step RT-PCR Kit, Ambion, Austin, TX, USA) with a 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA), although this test cannot differentiate between replication-competent and replication-incompetent virus. Primers and a minor groove binder (MGB) fluorescent probe specific to a conserved region of ORF7 were employed as described previously [60 (link)].

HIV viral sequencing using a nested PCR approach was performed on subject plasma samples at IIID, Murdoch University (Perth, WA, Australia), as previously described [61 (link)]. Briefly, HIV RNA was extracted from subject plasma samples using the Life Technologies MagMAX Viral RNA Isolation Kit, as per the manufacturer’s instructions. Two overlapping RT-PCRs were performed to cover the entire HIV genome. Resultant first round products were used as templates in two separate nested PCRs targeting the Gag, Pol and Nef genes. Standard bulk sequencing was carried-out using the Roche 454 Life Sciences GS-FLX platform. PCR products were quantified and equimolar pooled for each individual. Products were ligated to adaptors and clonally amplified on capture beads in water-in-oil emulsion micro-reactors, with the enriched products being sequenced on picotitre plates. Nucleotide data was collected and quality filtered using the Roche 454 software (default settings). Viral sequences were aligned to the HXB2 reference HIV clade B strain (GenBank accession number K03455) and a consensus sequence was generated for each subject using an in-house developed alignment tool (http://www.iiid.com.au/software/vgas).

Viral RNA was extracted and purified from ~10% (0.1 gram) stool suspension using MagMAX Viral RNA Isolation Kit (Life Technologies). Norovirus detection and noroviral load quantification was performed by using a quantitative, genogroup-specific, probe-based real-time reverse transcription–polymerase chain reaction (RT-qPCR) assay that targeted the junction between RNA-dependent RNA polymerase (RdRp) and viral protein 1 (VP1) genes of norovirus as previously described30 (link). Briefly, 10 μL of purified RNA was used as template in a 25-μL total reaction volume using SuperScript III Platinum One-Step qRT-PCR kit (Life Technologies). No template (negative) control was included in every run. To control for batch-to-batch variation, a positive control of known Ct value was included in each run. Assay cycle threshold (Ct) value was determined for each case to represent noroviral load. Higher Ct values indicated lower noroviral loads.

Noroviral RNA was extracted from ∼10% stool suspension using MagMAX Viral RNA Isolation Kit (Life Technologies). Purified viral RNA was first subjected to a GII.4-specific assay that amplified the complete VP1 gene using SuperScript III One-Step RT–PCR System (Life Technologies). Primers used were COG2F56 (link) and NoV-Capsid-R (5′-GAGCCRAGGACATCAGATGCC-3′). Samples tested negative for GII.4 norovirus were subjected to a separate round of cDNA synthesis step and PCR detection as we described previously52 (link). Briefly, viral RNA was converted into cDNA using a tagged oligo(dT) primer (5′-GACTGACTAGCTATCGGAGCATCG(T)₃₁-3′) and SuperScript III reverse transcriptase (Life Technologies). PCR of an ∼2.7 kb fragment covering complete VP1, VP2 and 3′ untranslated region of the norovirus genome was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific). Primers used were COG1F (for norovirus GI) or COG2F (for norovirus GII)56 (link) and Tag (5′-GACTGACTAGCTATCGGAGCATCG-3′; for both norovirus genogroups). PCR amplicons were purified using either ExoSAP-IT (Affymetrix) or QIAquick PCR Purification Kit (Qiagen). Sequencing was performed using the forward PCR primer. Norovirus genotype assignment was based on the partial VP1 gene using the norovirus genotyping tool (http://www.rivm.nl/mpf/norovirus/typingtool).