Magmax express 96

Viral RNA was purified from 200 µL of UTM by the MagMax Core Nucleic Acid Purification Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) in automated sample preparation workstation MagMAX Express 96 (Applied Biosystems), according to the manufacturer’s instructions.
Samples were tested by TaqPath™ COVID-19 CE-IVD RT-PCR Kit (Thermo Fisher) using the 7500HT Fast Real-Time PCR System (Applied Biosystems) according to manufacturer’s instructions. The kit is a multiplex assay that allows amplification of conserved regions in the S, N, and ORF1ab genes of the SARS-CoV-2 genome. Bacteriophage MS2 was used as an internal control to prevent false-negative results due to inhibition factors. Ct values were generated using the 7500 Software SDS 2.3 (Applied Biosystems).
Data analyses were performed by the Applied Biosystem^TM COVID-19 Interpretive Software, v.1.3 (Thermo Fisher Scientific, Waltham, MA, USA).

All PF samples were tested for the presence of SVA RNA by reverse-transcription real-time PCR (RT-rtPCR). RNA was extracted using a commercial extraction kit (Ambion MagMAX-96 viral RNA isolation kit; Life Technologies) and a magnetic particle processor (MagMAX Express-96 magnetic particle processor; Applied Biosystems), following the manufacturer’s guidelines. Although samples being assessed with RT-rtPCR are typically considered positive when the cycle threshold (Ct) values are 35.99 or lower and suspect when between 36 and 40 [16 (link)], we chose to classify any week as positive if at least one sample collected during that week had a Ct value under 40. Due to the decrease in sensitivity caused by aggregating multiple litters in one PF sample (due to a dilution effect) and since the prevalence of SVA-affected litters could be low (further decreasing sensitivity), suspect samples were treated as positives.

From each pooled urine sample 500 μl was added to MagMax lysis buffer (Applied Biosystems) and the RNA was extracted using the MagMax Viral RNA Isolation Kit (Cat No. AM1836) on the MagMax Express 96 automated extraction unit (Applied Biosystems). After extraction, RNA was stored at -80°C.

Swabs were hydrated in 400 µL of sterile PBS (pH 7.4) for five minutes at room temperature and transferred to swab extraction tube system (SETS; Cat. No. 3315568, Roche, IN, USA) tubes. SETS tubes were centrifuged at 6000 rpm for one minute to collect the elute, after which, inner SETS tubes and swabs were discarded. Tissues were homogenized in 500 µL of PBS with a sterile steel bead in two three-minute runs (icing in between) using a Geno Grinder 2000 (SPEX SamplePrep LLC, Metuchen, NJ, USA). A total of 100 µL of either swab eluate or homogenized tissue were aliquoted and used for DNA extraction; the remaining eluate or tissue homogenate volume was kept for viral titration. DNA was extracted from all swab and tissue samples using the MagMAX DNA Multi-sample Ultra kit (Applied Biosystems, Foster City, CA, USA) extraction protocol on a MagMAX Express-96 deep well magnetic particle processor (Applied Biosystems).

Nucleic acids from the same 300 individual mosquitoes used in pooling experiments were purified from 250 µL of supernatant to determine the prevalence and distribution of six representative viruses identified from HTS analysis. Supernatants were processed using the same modified MagMax Express-96 platform (Applied Biosystems) protocol as described above. Complementary DNA was prepared from TNA using SuperScript III (Invitrogen) and random hexamer primers. PCR screening primers were designed using assembled viral sequences obtained from HTS data (Table 2). All PCR assays were performed using an annealing temperature of 60 °C followed by 10 cycles, decreasing by 0.5 °C per cycle, and a final annealing temperature of 55 °C was maintained for a further 35 cycles. All PCR products of the anticipated size were confirmed using Sanger sequencing.

DNA was extracted from 100 mg of fecal content samples using the MagMAX DNA Multi-sample kit and the MagMAX Express-96 well magnetic particle processor (Thermofisher, Waltham, MA, USA) to achieve an automatic and standardized DNA extraction across samples. Briefly, fecal samples were mechanically homogenized with 2.8 mm ceramic beads (MoBio, Carlsbad, CA, USA; an additional 0.2 g of 0.1 mm glass beads were added), 100 μl of guanidine thiocyanate-EDTA-N-lauroyl sarcosine, and 800 μl of 200 mM NaPO₄. Samples were homogenized for 3 min at 300 rpm and were centrifuged 5 min at maximum speed. 200 μl of the extract was added to the MagMAX Express plate for further DNA extraction procedures according to manufacturer’s instructions. Isolated DNA was kept at − 20 °C (or − 80 °C for longer storage). Polymerase chain reaction (PCR) amplification of the variable 3 (V3) region of the 16S rRNA gene was performed as previously described [21 (link), 22 (link)]. Purified PCR products were sequenced using the Illumina MiSeq platform by the McMaster Genomics facility. Of note, we lost two samples during processing; one from baseline group and one from 4 weeks HSD group. The 16S rRNA samples metadata table is available in Additional file 1.

DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen) and run on the automated Qiacube platform (Qiagen) following the Purification of DNA from tissues protocol. RNA was extracted using the MagMAX-96 Viral RNA Isolation Kit (Thermo Fisher Scientific) on the MagMAX Express-96 (Thermo Fisher Scientific) magnetic bead processor.