To monitor the recovery performance of the SARS-CoV-2 RNA biomarker detection workflow, the bacteriophage Phi 6 was spiked into each sample (500 PFU.mL−1 final concentration) prior to sample concentration. PCR inhibition was monitored in the aqueous and particulate phases using a synthetic internal positive control (IPC) (VetMAX™ Xeno™ Internal Positive Control, A29762 and VetMAX™ Xeno™ Internal Positive Control - VIC™ Assay, A29767; ThermoFisher Scientific), which was added to the RT-qPCR mix following the instructions of the manufacturer. To determine PCR inhibition, IPC DNA was added to positive control wells (SARS-CoV-2 RNA, EVAg-GLOBAL material) and the mean Cq value ± standard deviation (n = 18) was used as reference point to determine if inhibition occurred in wastewater samples. The latter were considered to have PCR inhibition if the Cq values of the IPC exceeded the reference point by >2 Cq values (Ahmed et al., 2020d ). Each RT-qPCR reaction was run in triplicates, both for standards and microcosm samples. To minimize the risk of RT-qPCR contamination, RNA extraction and RT-qPCR analysis were performed in separate laboratories. Extraction blanks were added to each RNA extraction run and no template controls were added to each RT-qPCR plate.
Vetmax xeno internal positive control vic assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The VetMAX™ Xeno™ Internal Positive Control - VIC™ Assay is a laboratory product designed to be used as a control in diagnostic testing procedures. It serves as an internal positive control, providing a consistent and reliable reference point for the assessment of sample preparation and amplification efficiency.
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2 protocols using vetmax xeno internal positive control vic assay
1
SARS-CoV-2 RNA Biomarker Detection Workflow
2
qPCR Amplification of Purified DNA
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The VetMAX™-Plus qPCR Master Mix (ThermoFisher Scientific, Waltham, MA, USA) was used for DNA amplification. The PCR amplification mixture comprised 12.5 µL 2× qPCR Master Mix, 400 nM (final concentration) forward/reverse primers, 150 nM (final concentration) probe, 1 µL VetMAX™ Xeno™ Internal Positive Control-VIC™ Assay (ThermoFisher Scientific, Waltham, MA, USA), 2 µL template DNA, and nuclease-free water to make up a total volume of 25 µL per reaction.
Detection and amplification of purified DNA by qPCR was performed on the StepOnePlus™ Real-Time PCR System running StepOne Software v2.3 system (ThermoFisher Scientific, Waltham, MA, USA). The cycling conditions recommended by the manufacturer of the PCR reagents were used.
Detection and amplification of purified DNA by qPCR was performed on the StepOnePlus™ Real-Time PCR System running StepOne Software v2.3 system (ThermoFisher Scientific, Waltham, MA, USA). The cycling conditions recommended by the manufacturer of the PCR reagents were used.
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