Maxwell ht 96 gdna blood isolation system

Stool samples, reagent-only negative controls, and mock community positive controls (Zymo Research) were extracted using either the AllPrep PowerFecal DNA/RNA 96 Kit (Qiagen) or the Maxwell HT 96 gDNA Blood Isolation System (Promega) [27 (link)]. SARS-CoV-2 viral load was quantified as per CDC guidelines [28 ] using the 2019-nCoV N1 primer and probe set [28 ], as well as human RNaseP as an internal control. Each RT-qPCR reaction contained TaqPath™ 1-Step RT-qPCR Master Mix (Thermo Fisher), RNA template, the CDC N1 or RNaseP forward and reverse primers (IDT), probe, and RNase-free water to a total reaction volume of 10 μl. Viral copy numbers were quantified using N1 quantitative PCR (qPCR) standards (IDT) in tenfold dilutions to generate a standard curve. The assay was run in triplicate for each sample with three no-template control wells per 384 well plate.

The same nucleic acid extraction approach was applied to all sample types as previously described [31 (link)]. In brief, treated and untreated samples, reagent-only negative controls, and mock community positive controls (Zymo Research) were extracted using a protocol optimized for respiratory samples with a magnetic bead-based protocol using the Maxwell HT 96 gDNA Blood Isolation System (Promega) on a KingFisher Flex instrument. Briefly, cetyl trimethyl ammonium bromide (CTAB) is added to samples in individual Lysing Matrix E tubes (MP Biomedicals), incubated at 95°C for 5 minutes followed by beadbeating for three 30 second cycles at 7.0 m/s, incubated with proteinase K at 70°C for 10 minutes, 300 sample µL lysate collected, additional beadbeating for three 30 second cycles at 7.0 m/s with each cycle, and additional 300 sample µL lysate collected. Sample lysates are transferred to 96 well plates for binding, washing, and elution steps on the Kingfisher Flex sample purification system.

Stool samples, reagent-only negative controls, and mock community positive controls (Zymo Research) were extracted using either the AllPrep PowerFecal DNA/RNA 96 Kit (Qiagen) or the Maxwell HT 96 gDNA Blood Isolation System (Promega)^{53 (link)}. SARS-CoV-2 viral load was quantified as per CDC guidelines⁵⁴ using the 2019-nCoV N1 primer and probe set⁵⁴ , as well as human RNaseP as an internal control. Each RT-qPCR reaction contained TaqPath™ 1-Step RT-qPCR Master Mix (Thermo Fisher), RNA template, the CDC N1 or RNaseP forward and reverse primers (IDT), probe, and RNase-free water to a total reaction volume of 10 μl. Viral copy numbers were quantified using N1 quantitative PCR (qPCR) standards (IDT) in 10-fold dilutions to generate a standard curve. The assay was run in triplicate for each sample with three no-template control wells per 384 well plate.