M2000sp instrument

The Abbott Real-time SARS-CoV-2 assay (Abbott Molecular, Inc.) test was performed as described in the manufacturer’s instruction, which has received EUA from WHO and FDA^{19 ,22} . In this protocol pre-extraction sample inactivation was performed with a water bath at 56 °C for 30 min^{23 (link)}, after viral inactivation, nucleic acid extraction was performed from 0.5 ml VTM on the Abbott m2000 SP instrument and using the Abbott m2000 DNA Sample Preparation System according to the manufacturer’s recommendations. The amplification and detection were performed by Abbott m2000 RT-PCR instrument targeted to dual-target assay for the RdRp and N genes. The SARS-CoV-2 and IC-specific probes are each labeled with a different fluorophore, Carboxyfluorescein (FAM), Carboxy-X-rhodamine (ROX), and VIC P (Proprietary dye) for target and internal control detection, thus allowing for simultaneous detection of both amplified products¹⁹ .

The Abbott RealTime CMV assay is carried out on an Abbott m2000 system. It has two automated test procedures, for processing plasma and whole-blood specimens. Sample preparation and PCR assembly are performed on an Abbott m2000sp instrument. Real-time PCR amplification and detection and result reporting are performed on an Abbott m2000rt instrument. The test procedures for plasma and whole blood share the same reagents for sample preparation, PCR amplification, and detection but differ in their sample input and elution volumes. DNA is extracted from 0.5 ml of a plasma sample and eluted in a 70-μl eluate or from 0.3 ml of a whole-blood sample and eluted in a 110-μl eluate. Plasma samples can be processed by either extraction procedure, but WB samples can be extracted only by the WB extraction procedure.
For both procedures, the total PCR volume is 60 μl (35 μl eluate and 25 μl master mix). An internal control (IC) is mixed into the lysis reagent before the initiation of sample preparation and is added to each specimen, calibrator, and control as a control for extraction efficiency and to monitor PCR inhibition.

The cryopreserved samples were obtained during the initial hospital visit and were kept at −80 °C without thawing. PCR data for SARS-CoV-2 were accessible for all patients. Diagnosis of SARS-CoV-2 infection relied on the detection of SARS-CoV-2 DNA in nasal or oral swab samples confirmed by qPCR. The SARS-CoV-2 viral load was assessed using the Abbott RealTime SARS-CoV-2 qPCR assay on the m2000 RealTime platform, which includes automated DNA extraction by the m2000 sp instrument and real-time PCR by the m2000rt instrument (Abbott Laboratories in Abbott Park, IL, USA).

Fluorescence-activated cell sorting was performed on a BD FACS Aria II Cell Sorter (Becton Dickinson, Franklin Lakes, NJ), and data were analyzed using FACS DiVa software. Forward scatter (FSC) and side scatter (SSC) were used to identify human RBCs. HO was excited by a 407 nm violet laser and detected by a 440/40 filter. Thiazole orange was excited by a 488 nm blue laser and detected by a 530/30 filter. Infected cultures stained with TO only or HO only served as compensation controls. Infected RBCs containing single ring-stage parasites were gated and sorted from synchronous P. falciparum cultures.
Uninfected RBCs and single-ring iRBCs were individually collected into 100 µL of NucliSENS Lysis Buffer in 96 well plates and then stored at −80°C. Thawed samples were diluted in 1.9 mL of lysed whole human blood—a mixture of EDTA-anticoagulated human whole blood and NucliSENS Lysis Buffer at a volumetric ratio of 1:40 for nucleic acid extraction using the Abbott m2000sp instrument as described below.