Introduction
Emerging infections account for at least 15% of all human pathogens
(Petersen et al., 2018). To prevent the rapid spread of viruses,
particularly airborne transmitted viruses, rapid and accurate diagnostic
systems are essential. Point-of-care (POC) diagnostic platforms that
meet the ASSURED criteria from the WHO (Affordable, Sensitive, Specific,
User-friendly, Rapid and robust, Equipment-free, and Delivered) are of
special interest in this regard. These platforms can be performed
entirely in situ , from sample collection to final reading, in an
integrated manner (Iliescu et al., 2021).
According to the One Health approaches, SARS-CoV-2 and avian
metapneumovirus (aMPV) are two examples of RNA viruses of interest for
people and animals, respectively. Portable antigen tests can detect an
active infection, but they are less reliable than molecular tests (qPCR
or qRT-PCR as gold standard), especially in asymptomatic patients
(Schuit et al., 2021). Thus, to prevent silent SARS-CoV-2 outbreaks and
transmission, accurate and highly sensitive molecular systems are
needed. aMPV infection is considered one of the most economically
important upper respiratory tract diseases in poultry (Kabouni and
Lachhbeb, 2021). Early described as Turkey Rhinotracheitis (TRT) virus,
it mainly affects turkeys but causes the Swollen Head Syndrome (SHS)
disease in chickens as well. Different avian respiratory diseases have
very similar clinical signs to TRT, such as avian infectious
laryngotracheitis (ILT), avian infectious bronchitis (IB) or Newcastle
disease (ND). Therefore, a specific and sensitive diagnostic system is
essential to investigate its actual epidemiology in ongoing respiratory
outbreaks in farms.
Currently, the gold standard for molecular detection of many infections,
such as SARS-CoV-2 or aMPV, is based on qPCR techniques. However, this
method requires specialized training and expensive equipment, which can
limit its implementation in the field. Therefore, there is a growing
interest in developing robust and reliable molecular diagnostics
platforms that utilize the Reverse Transcriptase coupled to
Loop-mediated isothermal amplification (RT-LAMP) technique, as an
attractive and alternative point-of-care (POC) technique for the
detection of RNA viruses. LAMP protocols are based on nucleic acid
amplification under isothermal conditions, facilitated by a DNA
polymerase with strand displacement activity and a set of four to six
specific primers (Notomi et al., 2015). Detection can be achieved using
RT-LAMP procedures, as an endpoint by visualizing the amplification
products by DNA-agarose gel electrophoresis, or with the naked eye by
colorimetric detection. For example, detection of SARS-CoV-2 by RT-LAMP
can be achieved by using pH indicators, such as hydroxynaphthol blue
(Thompson and Lei, 2020; Juscamayta-López et al., 2021; Nawattanapaiboon
et al., 2021; Trassante et al., 2021; Raddatz et al., 2022), or metal
indicators that change colour depending on the concentration of free
Mg2+ ions (Goto et al., 2009). While these methods
have performance limitations, they have been already approved as usefulin vitro diagnostic (IVD) tools for large-scale screening.
Colloidal gold nanoparticles (AuNPs) have recently gained popularity as
an alternative POC-test based on colorimetric assays, which can use
either unlabelled AuNPs or DNA-functionalized AuNPs (DNA-nanoprobes) in
combination with a portable device (Sivakumar, 2021; Raddatz, 2022).
AuNPs possess unique Plasmon Resonance properties, which have already
demonstrated promising applications for colorimetric detection of
pathogens (Liu and Liu, 2017). Specific molecular detection with
DNA-nanoprobes relies on RNA or DNA hybridization, which causes
nanoprobe instability and a marked colour shift visible within 15
minutes (Carter et al., 2013; Sabela et al., 2017). In summary,
AuNPs-based colorimetric assays offer an attractive POC alternative for
rapid and sensitive molecular detection of pathogens.
In this study, we validate a POC tool based on RT-LAMP coupled to
specific DNA-nanoprobes for viral detection as a proof of concept. This
approach provides high sensitivity and accuracy levels close to RT-PCR
based diagnosis for SARS-CoV-2 or aMPV. Moreover, the combination of
these techniques makes our colorimetric system doubly reliable: RT-LAMP
amplification of the viral target with a set of 6 specific primers,
followed by its detection by specific-sequence DNA-nanoprobes.