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.