2.2. Sample Preparation, Nucleic-Acid Extraction, and Real-Time RT-PCR

Human nasopharyngeal swabs were collected using flocked-tip swabs and transported to the laboratory in a Copan universal transport medium (UTM-RT) system (Copan Italia, Brescia, Italy). Total nucleic acids were isolated from 190 µL of each human nasopharyngeal swab using a total nucleic acid isolation kit on a MagNa Pure Compact instrument (Roche Applied Science, Mannheim, Germany), according to the manufacturer’s instructions. An additional 5 µL of Equine herpesvirus 1 and Equine arteritis virus isolates were added to all samples for external DNA and RNA control and were detected in separate duplex PCR reactions with other targets [32,33].

All four HCoVs (229E, OC43, NL63, and HKU1) and all other respiratory viruses, including respiratory syncytial virus (RSV), human rhinoviruses (HRV), human metapneumovirus (HMPV), human bocavirus (HBoV), adenoviruses (AdVs), parainfluenza viruses 1-3 (PIV 1-3), enterovirus (EV) and influenza viruses A and B (Flu A-B) were detected by using one-step real-time RT-PCR assay in a Step-One Real-Time PCR system (Applied Biosystems, Carlsbad, CA) [34,35,36,37,38,39,40,41]. A total 5 µL of total nucleic acid was added to 15 µL of reaction mixture including 2 X Reaction Mix, SuperScript^® III RT/Platinum^®TaqMix (Invitrogen, Carlsbad, CA, USA). The cycling conditions were universal for all tested respiratory viruses: 20 min at 50 °C, 2 min at 95 °C, and 45 cycles of 15 s at 95 °C and 45 s at 60 °C.

From 133 affected live cattle (collected from 24 different cattle herds) with symptoms of respiratory disease, nasopharyngeal swab samples were collected into sterile swabs (Sigma Virocult^®, MW 951S, Leicester, UK). From 84 dead cattle with pneumonia and/or diarrhea originated from 76 different cattle herds, 10 cm³ lung tissue and/or feces samples were collected. About 1 cm³ of the samples was homogenized in dilution 1:10 in RPMI-1640 (Gibco, Life Technologies Inc., Grand Island, NY, USA) and stored at <−15 °C until testing. Total RNA was extracted from 140 µL of homogenate using a commercial kit for RNA extraction (QIAamp^® Viral RNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Individual samples were tested using a commercial real-time PCR method, detecting specific nucleic acids of seven different respiratory pathogens, including the detection of endogenous internal positive control (IPC) for controlling the efficiency of extraction and the absence of inhibitors in individual samples. Samples were tested on a 96-tube microplate. On each microplate, the positive controls for all tested pathogens were included. A commercial TaqMan^® real-time PCR kit for the detection of seven major ruminant pathogens (LSI VetMAX™ Screening Pack–Ruminants Respiratory Pathogens, LSI, Lissieu, France), which allows the simultaneous detection of the Micoplasma bovis, Histophilus somni, Pasteurella multocida, Mannheimia haemolytica, BCoV, bovine respiratory syncytial virus (BRSV), and Bovine parainfluenza 3 (PI-3) was used as previously described [27]. The amplification was performed using an Mx3005P real-time PCR machine (Stratagene, San Diego, CA, USA). The fluorescent signal was detected after each annealing, and the results were presented as a cycle threshold value for individual samples. Analysis of real-time amplification curves was performed using commercial thermal cycler system software; to determine fluorescence baselines, an “auto baseline” was used.