Samples were received by the onsite laboratory from the KCMC medical staff and inactivated using a recognized method [14 (link)] inside a flexible film negative-pressure isolator followed by RNA extraction and purification. Quantitative reverse-transcription PCR (qRT-PCR) assays targeting the L and NP genes were used to detect the presence of Zaire Ebola virus using Lightcycler RNA Master Hydrolysis reagents (Roche, Laval, PQ) according to manufacturer's instructions. Assays were performed on the Lightcycler Nano platform (Roche). Primary testing focused on EVD diagnosis using cut-off cycle threshold (CT) values for positive, equivocal, and negative of <36, 36.1–40, and >40, respectively. The lower the CT value, the higher the Ebola viral load and vice versa. The assays detected EVD at 10 genome equivalents (g.e.)/reaction. For differential diagnostics when requested, the laboratory offered qRT-PCR tests for Lassa virus [15 (link)] and Plasmodium species [16 (link)]. As an internal control for extraction and amplification procedures, MS2 phage was added to each sample as previously described [17 (link)].
Lightcycler nano platform
Manufactured by Roche
The Lightcycler Nano platform is a compact real-time PCR system designed for reliable and efficient nucleic acid amplification and analysis. It offers precise temperature control and optical detection capabilities to enable accurate and reproducible quantification of target sequences.
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Lab products found in correlation
2 protocols using lightcycler nano platform
1
Ebola Virus Detection in Clinical Samples
2
RT-qPCR Analysis of FaERF2 and FaACO1
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RT-qPCR was performed on a LightCycler ® Nano platform (Roche Diagnostics GmbH, Mannheim, Germany), using 2 µL cDNA in a system containing 1 µL forward and reverse primer (10 µM), 10 µL FastStart Essential DNA Green Master 2X (Roche), in a final volume of 20 µL. The following program was used: pre-incubation for 10 min at 95°C, followed by 45 cycles of 20 s at 95°C, 20 s at 60°C, and 20 s at 72°C. Gene-specific primer sequences were as follows: FaERF2 (Bombarely et al., 2010) , FaACO1 (forward: 5'-TGCTTTTGTTGCGAAATCAG-3'; reverse: 5'-ACCAAGTCCACTTCCAC CAG-3'). Primers specific for actin (Chai et al., 2011; Sun et al., 2013) and the intergenic region 26S-18S (Cumplido-Laso et al., 2012) were used as internal standards. Data obtained were analyzed using the qPCR instrument software version 1.0 (LightCycler Nano System, 2011) .
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