Magmax 96 ai nd viral rna isolation kit

Total RNA was extracted from specimens using MagMAX™ 96 AI/ND Viral RNA isolation kit (Thermo Fischer Scientific, Vilnius, Lithuania). Real‐time reverse transcription‐PCR (RT‐PCR) was performed to detect the M gene of NDV using OneStep RT‐PCR kit (Qiagen, Hilden, Germany) and previously published primers (Wise et al., 2004 (link)). Based on standard controls, a C_t value of 35 and below was regarded as positive. In order to assess whether the M gene‐positive samples contained lentogenic or mesogenic/velogenic NDV, all these samples were tested by RT‐PCR targeting the F gene of mesogenic/velogenic NDV APMV‐1 using previously published primers (Farkas et al., 2009 (link)). Any sample with a C_t value of 40 or below was regarded as positive.

Three highly pathogenic H5N1 avian influenza virus-containing cloacal or tracheal swab samples collected from wild birds were chosen because not only do these samples represent the typical sample received in the laboratory for wild-bird surveillance testing, but they also span the cycle threshold (Ct) range typically seen in positive samples (Table 1).
Viral RNA was extracted using the MagMAX-96 AI/ND Viral RNA Isolation Kit (ThermoFisher) using a KingFisher magnetic particle processor and 90 µL elution volume.

Total RNA was extracted from 50 μL of each CL or OP sample (prepared separately for each bird) using the MagMAX™-96 AI/ND Viral RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and eluted in 50 µL of elution buffer. To selectively deplete ribosomal RNAs (rRNAs) from the host (18S, 28S, and mitochondrial) and bacteria (16S/23S), 12 µL of the total RNAs were treated with an in-house RNaseH rRNA depletion protocol and purified using SPRI beads (Agencourt RNAClean XP Kit; Beckman Coulter Life Sciences, Indianapolis, IN, USA) as recently described [33 (link)].

The oropharyngeal (OP) and cloacal (CL) samples used in the current study were part of a consignment of samples from backyard chickens collected at live bird markets (LBMs) in Arusha, Dar es Salaam, Iringa, Mbeya, Morogoro and Tanga in Tanzania, during surveillance of the Newcastle disease virus (NDV) conducted between September 2018 and May 2019. From each bird, one OP and one CL was collected. During the sampling period, the flocks did not present overt clinical signs consistent with avian diseases, and their vaccination statuses or histories were not available. After collection using standard procedures [36 (link)], the samples were shipped to the Southeast Poultry Research Laboratory (SEPRL) in Athens, GA, USA for total RNA extraction using the MagMAX™-96 AI/ND Viral RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) as recently described [37 (link)]. Preparation of sequencing libraries (sequence-independent, single-primer amplification [38 (link)] and Nextera ^TM Flex protocols) and paired-end NGS (500-cycle MiSeq Reagent Kit v3) using the Illumina MiSeq platform were performed as previously described [39 (link)]. For the current study, 20 birds that were NDV-positive by real-time reverse transcription–polymerase chain reaction (rRT–PCR) were selected (n = 40 samples; one OP and one CL from each of the 20 birds).

From each sample-spotted FTA card, 24, 3-mm disks (i.e., 6 disks per spotted area) were punched out using sterile disposable biopsy punches (Robbins Instruments, USA) and incubated for 30 min at room temperature in 240 μL of nuclease-free TE buffer (10 mM Tris-HCl; 0.1 mM EDTA, pH 8.0) to elute nucleic acids. Total RNA was extracted from 100 μL of the TE eluate using MagMAX™-96 AI/ND Viral RNA Isolation Kit (Thermo Fisher Scientific, MA, USA) on an automated KingFisher Magnetic Particle Processor (Thermo Fisher, USA) following manufacturer's instructions. To selectively deplete abundant host-specific RNAs (18S, 28S and mitochondrial) and bacterial rRNAs (16S/23S), extracted RNAs were treated with an in-house RNaseH host rRNA depletion protocol we have recently described (38 (link)).

Virus RNA was extracted from cell supernatant or quail oropharyngeal swabs and quantitated (52 (link)). Viral RNA was isolated using the MagMAX-96 AI/ND viral RNA isolation kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. A one-step qPCR based on the avian influenza virus matrix gene as a surrogate of virus replication was carried out in a LightCycler 480 real-time PCR instrument (Roche Diagnostics, Rotkreuz, Switzerland) using a LightCycler 480 RNA master hydrolysis probe kit (Roche Life Science, Mannheim, Germany) in a final reaction volume of 20 µL. Each reaction mixture contained 1 μ LightCycler 480 probes master mix, 0.5 µM forward and reverse primers, 0.3 µM probe, and 5 µL of RNA. The qPCR cycling conditions ran at 61°C for 10 min and a denaturation step of 95°C for 30 s, followed by 45 cycles of amplification at 95°C for 10 s, 60°C for 20 s, and 72°C for 1 s, with a final cooling step at 40°C for 10 s. A standard curve was generated using 10-fold serial dilutions of a WF10 virus stock of known titer to correlate qPCR crossing point values with the amount of virus in each sample.