Onestep pcr inhibitor removal kit

RNA extraction was performed using a semi-automated method with magnetic silica beads (supplied by bioMerieux, Marcy l’Etoile, France). After an incubation step at room temperature for 20 min, 100 μL of magnetic silica beads were added, and, after a further 10 min incubation, an automated procedure was performed using a nucleic acid purification system (Auto-Pure96, All Sheng Instruments, Zhejiang, China) or a Nuclisens MiniMag system (bioMerieux, Marcy l’Etoile, France). The extracted nucleic acids were then purified from potential PCR inhibitors using the OneStep PCR Inhibitor Removal Kit (Zymo Research, CA, USA).

RNA was isolated according to manufacturer protocols using the Qiagen RNAeasy DSP FFPE Kit. In cases of melanotic samples, the Zymo Research OneStep PCR Inhibitor Removal Kit was used. RNA was quantified with the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Gene expression profiling was performed using the NanoString nCounter Human Immunology v2 Panel (CodeSet Only), and data were analyzed using nsolver^TM analysis software. Heatmaps were generated using R version 4.3.1 and the “pheatmap” package, which uses Euclidean distance as the similarity measure and clusters samples based on the ‘complete’ method [14 ]. PCA plots were generated using R version 4.3.1 and the “factoextra” package [15 ]. Clustering was performed via singular value decomposition.

Settled solids were obtained from 15 wastewater treatment plants across the United States (Table S2). Solids were collected from the primary clarifier or settled from a 24-h composited influent sample using Imhof cones. Samples were collected in sterile containers and transported to the lab. Samples from the Bay Area were processed immediately, while other samples were stored at −80°C until analysis (between 5 and 20 months). None of the samples stored at −80°C underwent a freeze-thaw cycle prior to the current work.
Solids were dewatered using centrifugation, and then an aliquot of the dewatered solids was set aside for dry-weight analysis. Solids were then suspended in a buffer (approximately 75 mg/mL), homogenized, and centrifuged. This suspension of solids in buffer was found to alleviate inhibition of RT-PCR (21 (link)). An aliquot of the supernatant was processed for total nucleic acid extraction using Chemagic 360 (Perkin Elmer). Nucleic acid preparations from wastewater samples are known to contain PCR inhibitors that interfere with their accurate quantification using PCR-based methods. Therefore, inhibitors were removed using the OneStep PCR inhibitor removal kit (Zymo Research; catalog no. D6035), yielding nucleic acids in 50 μL of eluant. These methods have been published in detail (42 (link)), and step-by-step protocols are available on protocols.io (43 , 44 ).

We extracted DNA from 30 salmon gut mucosa samples. For the 21 parasitized salmon, we also extracted DNA from their associated cestode wash and cestode body samples. In parallel, we extracted four mock community samples as positive controls, using ZymoBIOMICS microbial community standard (Zymo Research) as an input. This mock community comprises eight bacterial strains and two fungal strains that are not expected to be part of the salmon gut microbiome. We also extracted four extraction blanks (sterile water or extraction buffer) as negative controls. The positive and negative controls were all processed in the same manner as the rest of the samples. All extractions were performed using the DNeasy PowerLyzer PowerSoil kit (Qiagen) following the manufacturer’s protocol. Before PCR amplification, we treated each sample with the OneStep PCR inhibitor removal kit (Zymo Research).

Longitudinal half-leaf sections were first pulverized using three steel beads in a microcentrifuge tube with a FastPrep^®-24 Tissue Homogenizer (MP Biomedicals, Solon, OH, USA), at a speed of 4.0 m s^-1, repeated at 1 min intervals until the mixture resembled a fine powder. Next, DNA was extracted from a ~5.0 mg subsample of the pulverized tissue using an Invisorb^® Spin Tissue Mini Kit (Stratec Molecular, Berlin, Germany), according to the manufacturer’s protocol. The resulting eluent was purified using a OneStep^™ PCR Inhibitor Removal Kit (Zymo Research, Irvine, CA, USA). Prior to use in qPCR assays, the concentration of each DNA sample was measured using an Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA) and adjusted to reflect a standardized concentration of 10 ng μL^-1. When adding 2 μL of template DNA to later qPCR reactions, this resulted in a final concentration of 1 ng μL^-1 DNA template per total reaction volume (20 μL).

We suspended cloacal swabs, tracheal swabs, SIC, and large intestine contents (LIC) in 1–4 mL MEM as a 10% suspension. We extracted RNA by using GenCatch Viral RNA Miniprep Kit (Epoch Life Science, https://www.fishersci.com). We further processed samples containing fecal matter by using OneStep PCR Inhibitor Removal Kit (Zymo Research Corporation, https://www.zymoresearch.com).
We determined viral RNA titers by real-time reverse transcription-PCR (rRT-PCR), as reported previously (23 (link)). In brief, we amplified a 541-bp fragment of the M gene that covered the quantitative RT-PCR–amplified fragment. We designed 5′-CGCGTAATCGTGTGATCTATGT-3′ and 5′-CCGGCCTTTGAAGTGGTTAT-3′ primers according to the sequence of a strain from the United States, Illinois121/2014 (GenBank accession no. KJ481931). We purified the PCR products by using a QIAquick PCR Purification Kit (QIAGEN Inc., https://www.qiagen.com), sequenced, and then used these as the template to construct a quantitative RT-PCR standard curve. The detection limit of the rRT-PCR was 10 genomic equivalents (GEs)/reaction, which corresponded to 4.6 log₁₀ GE/mL of PDCoV in cloacal and tracheal samples.

Soft tissue samples were extracted with either the QIAamp® DNA Mini Kit or QIAcube HT kits (QIAGEN, Inc.) using the manufacturer’s protocol, or the PrepMan Ultra reagent. DNA extraction from soft tissue samples using PrepMan was performed by wiping tissue surfaces for 30 s with a sterile cotton swab. The swab was then added to 150 μl of PrepMan Ultra reagent, vortexed for 15 s, and incubated for 5 min at 95 °C. PCR inhibitors were removed by adding the PrepMan/sample lysate to a column using the OneStep™ PCR Inhibitor Removal Kit (Zymo Research) per manufacturer’s protocol. FFPE samples were extracted using a modified FFPE extraction protocol from the QIAamp DNA Mini and Blood Mini Handbook (see supplementary information for details). Blood sample extraction was performed using the QIAcube HT kit. Fecal samples were extracted with the QIAamp® DNA Stool Mini Kit (QIAGEN, Inc.) or the PrepMan rapid-extraction protocol. DNA was extracted from fecal samples using PrepMan by adding 100 mg of feces to 150 μl of PrepMan Ultra reagent and diluted 1:10 with nuclease free water after the PCR inhibition removal step. Mock tiger bone wine DNA extraction was performed using a modified FavorPrep Stool DNA Isolation Mini Kit protocol (FAVORGEN Biotech Corp.) (the bead beating step was not performed; see supplementary information for protocol details).