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.