Discussion

In this study, we have developed and fully validated a method based on RT-LAMP amplification combined with specific DNA-nanoprobes for the detection of two RNA viruses, using SARS-CoV-2 and aMPV samples as a proof of concept for POC diagnosis of emerging respiratory pathogens. We suggest that the combination for RT-LAMP procedures, using three pairs of specific oligonucleotides, and DNA-nanoprobes targeting discrete regions of the viral genome reach both sensitivity and specificity levels comparable to gold standard procedures based on qRT-PCR methods. Therefore, a RT-LAMP/DNA nanoprobe-based device could be easily implemented as a cost-effective and reliable system widely applied for the screening of many diseases and viruses in situ , including human and avian viruses (Padzil et al., 2022)
The high sensitivity levels for LAMP are one of its main virtues as a potential alternative for molecular detection as POC devices, notably in developing countries or rural areas without basic health care infrastructures. However, some drawbacks of this technique have been widely reported, including potential cross-contamination (Tomita et al., 2008; Morris et al., 2015; Bao et al., 2020). To avoid this, detection kits for RT-LAMP have been improved by adding an UDG enzyme (Lai et al., 2022). This enzyme promotes the degradation of uracil-nucleotides on pre-amplified products but without any effect on the original template. In our study, the usefulness of this strategy has been successfully attained since the number of false positive results was virtually reduced to 0% using field samples.
As a drawback of the LAMP technique as a potential POC device concerns the read-out procedure to detect and discriminate positive over negative samples. Various methods have been described so far that do not require any equipment, enabling diagnosis by the naked eye. One of the first approaches relies on the turbidity measurement at end-point LAMP amplification. In fact, as the reaction progresses, magnesium pyrophosphate is produced and precipitates in the amplification of positive samples (Mori et al., 2001). However, in order to achieve a noticeable precipitate, this strategy relies on a high amplification efficiency and a substantial product yield. Otherwise, samples with low viral loads may be categorized as false negatives. Other LAMP systems use colorimetric indicators, such as hydroxynaphthol blue, a metal indicator that turns from violet to blue as free Mg2+concentration decreases when positive samples are successfully amplified. Nevertheless, this method has very low sensitivity levels, approximately 10-100ng of RNA (Hongjaisee et al., 2021), and low specificity values, around 80%, compared to gold standard qRT-PCR (Prakash et al., 2023). Phenol- or cresol-red are also pH indicators used as a read-out for LAMP-based diagnosis (Huang et al., 2020). These pH indicators can detect the release of protons during DNA synthesis, resulting in a color shift from pink to yellow in positive samples. While Raddatz et al. improved this system for SARS-CoV-2 detection, the slight pH changes registered during LAMP amplification may be one of the major limitations for its use in POC testing (Raddatz et al., 2022). This constraint can become particularly problematic when working with raw samples. In general, LAMP-based diagnostic technologies may exhibit lower specificity levels, leading to increased uncertainty in distinguishing false positive results. In contrast to these technologies, the colorimetric system validated in this study provides a highly sequence-specific read-out. This is because the DNA-nanoprobes we have designed for detection of either SARS-CoV-2 or aMPV can readily and specifically bind to those 100% identical viral RNA sequences upon undergoing RT-LAMP amplification.
Fluorescent-based LAMP has been also reported as an alternative for qPCR-based detection systems. For instance, it has been recently described a multiplexed (mLAMP) that is able to identify three different pathogens in one single reaction tube (Fan et al., 2022). While the specificity, sensitivity, and accuracy of this approach are comparable to PCR-based procedures, its readout based on fluorescence at different wavelengths requires more complex and costly equipment compared to the colorimetric developed in our study. Furthermore, this fluorescent-based LAMP procedure exhibits a sensitivity that is 10-fold lower (LOD > 500 copies/reaction) compared to our detection system. Nevertheless, a fluorescent-based LAMP for avian influenza virus detection has demonstrated impressive sensitivity (LOD>10 copies/reaction) (Padzil et al., 2022). Regardless, it is worth noting that, additionally, these systems require fluorescent-labeled primers, resulting in increased costs.
As far as the usefulness of AuNPs is concerned, although showing low LOD values, recent studies describe some detection systems based on its combination with the RT-LAMP technique in lateral flow biosensors (Zhu et al., 2020; Chen et al., 2021). Besides, a singular lab-on-a-chip system for SARS-CoV-2 detection based on the combination of LAMP reactions and colorimetric detection by AuNPs upon UV radiation has been recently developed (Sivakumar et al., 2021). In this study, the researchers employ unlabeled AuNPs that are synthesized in situon a portable device, instead of DNA-nanoprobes, to detect LAMP amplicons. Of note, this promising system has been just assayed using a synthetic plasmid DNA encoding the envelope (E) gene from SARS-CoV-2, whereas our detection system has been validated using a substantial number of real patient samples, specifically 140 samples from individuals with SARS-CoV-2. Although, it is not possible to compare their performance in terms of specificity and accuracy, both systems are able to detect LAMP amplicons by the naked eye within 45 min after LAMP reaction. Nonetheless, the use of unlabeled AuNPs and biosensors designed to target labeled LAMP primers (Zhu et al., 2020; Chen et al., 2021) may lead to a reduction in the specificity of colorimetric detection. Our system is based on sequence-specific colorimetric detection, that not only practically ensures no false positive results upon LAMP reaction, but also avoids detection of nonspecific amplifications or artifacts due to the formation of primer dimers.
Regarding the diagnosis of aMPV infection in poultry, while previous studies have developed diagnostic methods for other avian viruses (Padzil et al., 2022), this is the first study describing the development of an RT-LAMP for its molecular detection. The clinical diagnosis of aMPV in field conditions is not easy due to the occurrence of co-infections in animals. Currently, RT-PCR and qRT-PCR tests have become the established gold standard methods in reference laboratories for diagnosing active infections. Indeed, primer sequences for the RT-PCR have been designed for specific detection of the F, M, N and G genes (Ferreira et al., 2009; Kariithi et al., 2022; Wang et al., 2022). For instance, since the gene G shows the highest variability between subtypes it is the region most widely used at aMPV genotyping.
In this study, the usefulness of the F gene as a target for aMPV detection by RT-LAMP is based on its high conservation degree in the reference strains of aMPV subtypes A and B. A recent study supports these subtypes, specially aMPV-B, as the most prevalent not only in Spain but also in many European countries (Mescolini et al., 2021). We also performed different LAMP primer designs targeting the gene M but with lower sensitivity and specificity values using field samples (not shown). A highly desirable approach would be the development of a multiplexed LAMP strategy for the detection of viral respiratory co-infections and/or those that exhibit similar clinical signs to aMPV infection, irrespective of the readout device used.