Application of RT-LAMP and LAMP-sequencing for SARS-CoV-2 testing
With its good sensitivity for samples up to CT ≈ 30, the colorimetric RT-LAMP assay has several advantages: It is fast, inexpensive, and it can be evaluated without any equipment. RT-LAMP reactions also appear to be less sensitive to contaminants in the samples than RT-qPCR, but care has to be taken that the samples used do not alter the pH as the colorimetric RT-LAMP assay is performed under conditions of weak pH buffering. Some clinical samples contain contaminants that can lead to acidification of the reaction independent of the presence of a template RNA if too much sample is added. Diagnostic RT-qPCR tests usually include a technical internal control, i.e., another RNA species, which is spiked into all samples and which is detected independent of the gene of interest to safeguard against the possibility of a general reaction failure within a sample tube. It would be desirable to have a similar precaution for the RT-LAMP assay. A multiplexed fluorescence readout might provide this (34) but comes at the expense of the simplicity of a colorimetric readout.
Our particular implementation of deep sequencing to analyze many RT-LAMP reactions simultaneously uses two sets of barcoded primers and is fully scalable so that, in one sequencing run, many thousands of LAMP reactions can be quantitatively analyzed for the presence of viral genomic sequences. Although we used Illumina dye sequencing, more scalable sequencing technologies, such as Oxford Nanopore Technologies sequencing, could be used for amplicon sequencing and counting (37). The workflow shown here uses LAMP-sequencing as a validation and backup procedure to double check the results of the colorimetric RT-LAMP assay. However, LAMP-sequencing could also facilitate scale-up of the workflow for direct analysis of many thousands of samples in an efficient manner, provided that an infrastructure is established that allows the collection of such samples. Thus, LAMP-sequencing could become an important part of workflows for routine testing of large populations.
Schmid-Burgk et al. (38) proposed decentralized RT-LAMP assays using combinatorial primer barcoding and centralized mass analysis of RT-LAMP products by next-generation sequencing as a means to scale-up testing. Although this poses additional challenges in generating the individualized RT-LAMP assay reagents, it would simplify sample handling on the analytical side and it can be easily combined with the barcoding strategy shown here.
There are several limitations to our study. We used surplus RNA sample material from a diagnostic laboratory rather than newly collected clinical samples. The criteria for testing individuals may have influenced cohort characteristics and hence our findings. It is not clear yet how well viral load as indicated by CT values from RT-qPCR assays informs about the degree of infectivity of an individual with a SARS-CoV-2 infection. Therefore, we cannot say how our findings on the sensitivity of the RT-LAMP assay in comparison to RT-qPCR would translate into sensitivity for detecting infectious individuals who are shedding SARS-CoV-2 virus. Moreover, the measured viral load does not indicate the course of a SARS-CoV-2 infection, as even individuals with a very low measured viral load can still develop severe symptoms of COVID-19 disease. This may be, in part, because the viral load in a clinical sample taken from a specific site such as the pharynx is not representative of the overall viral burden that an infected individual carries.
We used LAMP-sequencing to validate the RT-LAMP assay results and did not use it as a diagnostic tool. LAMP-sequencing is dependent on the sensitivity of the RT-LAMP reaction as it cannot detect false negative results caused by a failure of the RT-LAMP assay to amplify viral RNA. Also, reagents such as the primer sets for the RT-LAMP assay may be subject to production-dependent quality fluctuations. Therefore, all reagents must be precisely validated (batch control) before using an RT-LAMP assay diagnostically.
Application of the RT-LAMP assay has great potential, even more so as more sensitive primer sets become available. The RT-LAMP assay and LAMP-sequencing could offer scalable testing that would be difficult to achieve with conventional RT-qPCR–based tests. For example, the RT-LAMP assay could be used for regular testing of a whole workforce or in sentinel testing, ideally combined with simplified sample collection, e.g., in the form of saliva samples. The RT-LAMP assay and LAMP-sequencing extend the range of available test methods and complement individual tests and pooled tests based on RT-qPCR (39) with a faster, simpler, and potentially more cost-effective test method.