2.2 Nucleic acid extraction
Nucleic acid extraction was performed in biosafety level 3 (BSL-3)
laboratories. Aliquots (400 µL) of the milk samples collected from each
cow were subjected to extraction and purification using the QIAsymphony
DSP Virus/Pathogen Midi Kit (Qiagen, Hilden, Germany) and processed
using the QIAsymphony automated system (Qiagen) according to the
manufacturer’s instructions, eluted in 60 µL, and stored at –80°C until
use.
Nucleic acid was extracted from nasal swabs as follows: 200 µL aliquots
of Universal Viral Transport Medium (UTM) (Copan) were used for nucleic
acid extraction and purification with the KingFisher™ Flex system
(Thermo Fisher Scientific) using the MVP_2Wash_200_Flex program
according to the manufacturer’s instructions. The extracted RNA was
eluted in a final volume of 50 µL and stored at –80°C until use.
SARS-CoV-2 RT-qPCR was performed in BSL-2 labs using the TaqPath
COVID-19 CE-IVD RT-PCR Kit (Thermo Fisher Scientific), which
simultaneously amplifies three viral targets, the ORF1ab gene (FAM), N
protein (VIC), and S protein (ABY); MS2 phage (JUN) is detected as the
internal positive control. The amplification was carried out in a final
volume of 25 μL, which included 5 μL of template, TaqPath 1-Step
Multiplex Master Mix (4×), and COVID-19 Real-time PCR assay Multiplex,
which contains probes and specific primer sets for different SARS-CoV-2
and internal control genomic regions. Each experiment also included
TaqPath COVID-19 IVT RNA as a positive control and a negative control.
The thermal cycling conditions consisted of an initial Uracil-DNA
glycosylase (UNG) incubation step at 25°C for 2 min, reverse
transcription at 53°C for 10 min, and an initial denaturation and enzyme
activation step at 95°C for 2 min, followed by 40 cycles of denaturation
at 95°C for 3 sec, and annealing/extension at 60°C for 30 sec. RT-qPCR
was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems,
Foster City, CA, USA).