A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples

We read with great interest the article by Dao Thi et al in which the performance of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay were compared with that of real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for detecting SARS-CoV-2 nucleic acid (1). Currently RT-PCR coupled with respiratory swab specimens in universal transport media (UTM) is the most widely used testing method for the diagnosis of SARS-CoV-2 infection. In the clinical setting repeat sampling in the patient often aids in diagnosis and monitoring of COVID-19. Hence increased utilization of laboratory resources such as PCR reagents, flocked swabs and UTM during this pandemic have caused supply shortage of these heavily used consumables. Recent deployment of novel testing strategies using alternative specimen types had generated some exciting findings. Multiple studies had previous proposed the use of saliva for COVID-19 diagnosis (2). A recent study (3) suggested saliva collected in patients with acute SARS-CoV-2 infection can substitute nasopharyngeal swabs with very high diagnostic concordance. There were high variations in RNase P gene Ct values in nasopharyngeal swabs than that in the paired saliva samples (4), indicating a greater degree of inconsistency in the former. This highlights pre-analytical variability could be introduced during the sampling of naso/oropharyngeal swabs although this procedure is usually conducted by trained medical personnel.

Recently saliva specimen was also reported to serve as a good alternative specimen for mass screening of SARS-CoV-2 in asymptomatic individuals (5). In view of the ongoing pandemic situation, diversifying testing for diagnostic and screening purposes using saliva which is not constrained by the availability of the flocked swabs and UTM, may ease the bottleneck of swab-based tests. Self-collection of saliva, a self-executed non-invasive process can also avoid close physical contact between healthcare personnel and patients. This may reduce risk of acquiring infections and utilization of personal protective equipment among the healthcare workers. However, compliance to the standardized saliva collection protocol which is important to ensure the downstream molecular test’s sensitivity is not compromised is difficult in children. More studies are also needed to demonstrate the diagnostic values of saliva sample in patients with mainly lower respiratory tract infections or pneumonia.

Notably, high concordance in testing results between real-time RT-PCR and RT-LAMP tests on saliva specimens in asymptomatic individuals (5). Although both of these techniques rely on amplification of nucleic acid, they leverage on different assay designs fundamentally. With multiple sets of primers targeting the adjacent regions of a specific viral target sequence, RT-LAMP is performed at a constant temperature with spectrophoto/colorimetric detection of its amplification products without the need of a thermal cycler. Commercial RT-LAMP assays usually have a slightly faster machine turnaround time and are scalable for high throughput testing. Using non-PCR methods for SARS-CoV-2 assays will free up thermal cyclers for a molecular laboratory to use for other routine assays.

At the time of writing this comment, there were 7 molecular diagnostic tests for SARS-CoV-2 leveraging on RT-LAMP technology that had received emergency use of authorization from the US Food and Drug Administration (FDA) (6). In our opinion, developing new assays by combining the strengths of orthogonal technologies and use of non-swab specimens such as saliva had helped the world to create diagnostic short-cuts during the current global crisis.

Acknowledgements
The authors thank Dr Roland Jureen and Dr Karen Mei-Ling Tan from the Department of Laboratory Medicine, National University Hospital Singapore, for their comments to this manuscript.

Author contributions
Kok-Siong Poon wrote the first draft. Nancy Tee critically revised the manuscript.

Conflict of Interest
The authors have no conflict of interest to declare.

References
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6. Food and Drug Administration. In vitro diagnostics EUAs. Available at: https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-em.... Accessed November 19, 2020.