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.