Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance

Abstract

Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/μL of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection of 39 copies/mL. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infection rates in Massachusetts and nationwide.

Improved visual detection of DNA amplification using pyridylazophenol metal sensing dyes

“…As a demonstration of PAPS-based detection, we applied it for detecting synthetic SARS-CoV-2 RNA by RT-LAMP. We performed 24 reactions with input of approximately 10 copies of viral RNA per reaction, which is below the limit of detection of a commercially available kit based on a pH-dependent dye (~50 copies, SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit, NEB #E2019 42 , 43 ), in order to observe both positive and negative reactions (Fig. 5 ).…”

Development of a Novel Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Monkeypox Virus Infections

“…Interestingly, mpox DNA can be found in the saliva of infected cases by using qPCR [ 43 ]. Previous reports showed that unpurified SARS-CoV-2 in saliva samples can be detected with no RNA extraction step via a RT-LAMP assay [ 44 , 45 ]. As the reaction temperature of our LAMP assay is 65 °C and mpox virus can be inactivated at 60 °C in under 15 min [ 46 ], this LAMP assay can theoretically be used to detect mpox from crude clinical saliva samples without prior DNA extraction.…”

Overcoming variant mutation-related impacts on viral sequencing and detection methodologies

“…For clinical samples, RNA extractions were performed as described ( 22 ) following NEB’s protocol for RNA extraction from saliva utilizing the Monarch Total RNA Miniprep Kit (T2010). The final total RNA was eluted in either 50 or 100 μl of nuclease free water prior to storage at –20°C for less than 1 week or –80°C for long term storage.…”