Viral loads in OP and CL swabs were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). RNA was extracted from swabs using the MagMAX96 Viral RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) and the KingFisher Flex Magnetic Particle Processing System (Thermo Fisher Scientific), with an additional wash step to remove inhibitors (Das et al., 2009 (link)). The qRT- PCR for AIV detection was conducted based on the standard USDA M gene AIV qRT- PCR procedure (Spackman et al., 2002 (link)) using an Applied Biosystems® 7500 Fast Real- Time PCR system (Thermo Fisher Scientific). Cycle threshold (Ct) values were determined by the 7500 Fast Software v2.3. For relative quantification, Ct values were converted to titer equivalents based on the standard curve method (Larionov et al., 2005 (link)). Values were established from ten-fold dilutions of the same titrated stock of the virus used to challenge the birds. The limit of detection was determined to be 0.8Log10 per reaction.
Kingfisher flex magnetic particle processing system
Manufactured by Thermo Fisher Scientific
The KingFisher Flex Magnetic Particle Processing System is a lab equipment designed for automated nucleic acid purification and sample preparation. It utilizes magnetic particles to capture, wash, and elute target biomolecules from a variety of sample types. The system provides efficient and reproducible sample processing with minimal hands-on time.
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2 protocols using kingfisher flex magnetic particle processing system
1
Quantitative Real-Time RT-PCR for Avian Influenza Viral Loads
2
Quantitative RT-PCR for Avian Influenza
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RNA was extracted from a total of 45 samples for each combination of collection device and sample location using the MagMax96 Viral RNA Isolation Kit System (Thermo Fisher Scientific) and the KingFisher Flex Magnetic Particle Processing System (Thermo Fisher Scientific). The manufacturer's instruction was followed with the addition of an additional wash step to remove inhibitors (Das, Spackman, Pantin‐Jackwood, & Suarez, 2009 ). qRT‐PCR was performed based on the standard USDA M gene AIV qRT‐PCR procedure (Spackman et al., 2002 ) using an Applied Biosystems® 7,500 Fast Real‐Time PCR system (Thermo Fisher Scientific). Cycle threshold (Ct) values were calculated by the 7,500 Fast Software v2.3 (Applied Biosystems). For relative quantification, cycle threshold (Ct) values obtained from qRT‐PCR were converted to titre equivalents based on the titre of the viruses, each of which was used as qRT‐PCR standards for the appropriate samples. The limit of detection (LOD) or the cut‐off value was determined based on the endpoint when the qRT‐PCR assay did not detect any signal in the RNA from serially diluted standard viruses.
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