Materials and Methods

Sample collection

Retrospective 140 pharyngeal swabs SARS-CoV-2 samples (denoted as S1 to S140) from both symptomatic and asymptomatic patients were provided in inactivating transport media by different Spanish healthcare agents. The sample collection was conducted during the sixth wave of COVID-19 in December 2021 in Spain, which was associated with the Omicron variant spread. Out of the collected samples, 72 tested positive for SARS-CoV-2, while 68 tested negatives. For this study, the sample processing was performed under BSL2 standard operating procedures at BioAssays (Certification n. CS17817 for health centers, services, and establishments by the Health Department of the Autonomous Community of Madrid). TaqPath™ COVID-19 CE-IVD RT-PCR Kit (ThermoFisher Scientific, USA) was used for confirmation. As a negative control, purified RNA from the human coronavirus 229E (hCoV-229E, GenBank Access Number: NC_002645.1) was used.
For aMPV, the “Centro de Sanidad Avícola de Cataluña y Aragón” (CESAC) kindly provided viral RNA from 50 samples (15 upper respiratory tract swabs and 35 tracheal tissue samples), denoted as M1 to M50, collected from 34 chickens and 16 turkeys (Table S1). Among the 50 samples, 33 were diagnosed as aMPV-positive using a qRT-PCR screening test targeting the Small Hydrophobic (SH) gene (Mescolini et al., 2021). Additionally, 17 samples that tested negative for aMPV but positive for other respiratory viruses affecting poultry, such as Infectious Bronchitis Virus (IBV) and Infectious Laryngotracheitis Virus (ILTV), were also analysed. Live attenuated vaccine NOBILIS® ND CLONE 30 (Merck Sharp and Dohme) was used as positive control for Newcastle Disease Virus (NDV). To optimize the diagnostic system for aMPV, the live-attenuated vaccine HIPRAVIAR SHS (strain 1062 from HIPRA S.A., Spain) available in vials with lyophilized suspension ranging from 102.4 to 104.4 TCID50 in 1 mL, was used as a positive control.

RNA extraction from clinical samples

Human pharyngeal swabs were extracted using the automatized robot Chemagic 360/96 RodHead and Chemagic Viral DNA/RNA 300 Kit H96 purification kit (Chemagen Technology, Perkin Elmer). HIPRAVIAR-SHS vial was reconstituted in 1 mL of phosphate buffer saline (PBS) and RNA was isolated using GeneJET RNA Purification Kit (ThermoScientific), following the manufacture’s recommendations.

RT-LAMP and qRT-LAMP amplifications

Purified viral RNA was amplified by one-step RT-LAMP or qRT-LAMP using WarmStart RTx Reverse Transcriptase and Bst 2.0 WarmStart® DNA Polymerase (New England Biolabs). Optimization of RT-LAMP reaction was performed in 25 µL by mixing 12.5 µL of WarmStart MasterMix (WarmStart® Fluorescent LAMP/RT-LAMP Kit with Uracil-DNA Glycosylase (UDG) (New England Biolabs, USA), 1 µL of each primer set (optimization from 0.8 to 1.6 µM internal FIP/BIP primers; 0.1 to 0.4 µM outer F3/B3 primers, and 0.2 to 0.6 µM loop LF/LB primers), 20 U Ribonuclease Inhibitor (NZYtech, Portugal) and 5 µL of purified RNA. Additionally, for qRT-LAMP, 0.5 µL LAMP Fluorescent Dye were added. Reaction mix was prepared at room temperature to allow UDG activity, then incubated at 60-65oC for 15 to 60 min for retrotranscription and Bst 2.0 amplification, followed by enzyme inactivation at 80oC for 5 min. Fluorescence signal was monitored along incubation in a QuantStudio™ 5 Real-Time PCR System (ThermoScientific), adding an end-point melt curve step to verify the specificity of the amplification. Alternatively, end point amplification was visualized in a 2% TAE-agarose gel electrophoresis.
To assess the specificity of the method, hCoV-229E RNA was used. Similarly, IBV and ILTV-positive samples, were underwent same procedures as the aMPV samples. The primer sets employed for the detection of SARS-CoV-2, targeting the nucleocapsid (N) and envelope (E), as well as the primer set for aMPV, targeting the fusion protein gene (F), are detailed in Table S2.

Oligonucleotide probes

To design the specific pangenotypic oligonucleotide probes for either SARS-CoV-2 or aMPV, we conducted CLUSTAL Omega alignments (https://www.ebi.ac.uk/Tools/msa/clustalo/) using the reference sequences (GenBank Accession Number: MT121215.1) and (GenBank Accession Number: NC_039231.1), respectively. To check system specificity, hCoV-229E genome was used. Then, 20 nt-long sequences were selected from the conserved regions of N and E genes (SARS-CoV-2) or F gene (aMPV). The designed probes were analysed for their secondary structure using the online RNAfold server (Vienna RNA Web Service; Gruber et al., 2008). Oligonucleotide probes with higher Gibbs free energy (ΔG0) values were chosen and synthetised. All oligonucleotide probes used in this study were synthesized by Sigma-Aldrich (Merck, USA) and included a 5’-Thiol modification.

Gold nanoparticles functionalization

Gold nanoparticles (AuNPs), 20nm, in citrate buffer solution (6.8x1011nanoparticles/mL) were purchased from Nanovex Biotechnologies (Spain). A solution of 6-mercapto-1-hexanol (MCH) and DTT-reduced thiol SARS-CoV-2 or aMPV probe-oligonucleotides (ratio 10:90, final probe concentration 2.5 µM) were incubated with 1 mL of AuNPs for 16h. Then, the mixture was salt aged up to 1.3 M NaCl, as previously described by Hurst et al. (2008), with a few modifications. Briefly, oligonucleotide probe/AuNPs mixture was incubated with 0.2 M NaCl for 2 h at room temperature, then 0.01% Tween-20 was added, and finally subjected to vacuum centrifugation to increase final salt concentration. Functionalized AuNPs with probe-oligonucleotides (DNA-nanoprobes) were washed twice with 10 mM PBS (pH 7.5) by centrifugation at 10,000 rpm for 15 min (MiniSpin Plus G, Eppendorf, Germany) at RT to remove unbound oligonucleotides. Finally, DNA-nanoprobes were resuspended in 10 mM PBS and conserved at 4oC until further use.

Detection by DNA-nanoprobes

To optimize colorimetric detection, 1.5 µL SARS-CoV-2- or aMPV-nanoprobes were incubated with 5 µL of RT-LAMP products in a 25 mM pH7.5 Tris-buffered reaction solution. For SARS-N nanoprobe, reaction buffer was supplemented with 3 M NaCl, 10 mM MgCl2, and 0.01% Tween-20, whereas for aMPV-F2 nanoprobe, it contained 2 M NaCl, 22 mM MgCl2, and 0.01% Tween-20. Sterile water was included as a negative control. Reaction was carried out on a thermal block at 37oC for 90 min. Absorbance intensity at 400-800 nm wavelength range was measured at different time points (15, 30, 45, 60 and 90 min) using a NanoDrop 2000 Spectrophotmeter (ThermoFisher Scientific), or at 90 minutes using a Multiskan FC plate reader equipped with a 540 nm filter (ThermoFisher Scientific). Based on visual screening of detection assays, we found that the most distinct contrast between the positive and negative samples was determined at 1:3 (v/v) ratio of LAMP products to DNA-nanoprobe.