A simple direct RT-LAMP SARS-CoV-2 saliva diagnostic

Abstract

Widespread, frequent testing is essential for curbing the ongoing COVID-19 pandemic. Because its simplicity makes it ideal for widely distributed, high throughput testing, RT-LAMP provides an attractive alternative to RT-qPCR. However, most RT-LAMP protocols require the purification of RNA, a complex and low-throughput bottleneck that has often been subject to reagent supply shortages. Here, we report an optimized RT-LAMP-based SARS-CoV-2 diagnostic protocol for saliva and swab samples. In the protocol we replace RNA purification with a simple sample preparation step using a widely available chelating agent, as well as optimize key protocol parameters. When tested on clinical swab and saliva samples, this assay achieves a limit of detection of 105 viral genomes per ml, with sensitivity close to 90% and specificity close to 100%, and takes 45 minutes from sample collection to result, making it well suited for a COVID-19 surveillance program.

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread

“…After these modifications, RT-LAMP achieved 77.2% overall clinical sensitivity and 97% specificity, with 93.2% sensitivity in saliva containing at least 10 2 viral copies/µL (Kappa = 0.895), which is on par or more sensitive compared to most of the direct RT-LAMP approaches reported so far [8,15,[25][26][27][28][29][30][31][32][33]. Moreover, the high agreement (>97.6%) observed between color interpretation and specific amplification plots in rtRT-LAMP demonstrates reliability on end-point color readout if real-time analysis is not possible.…”

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

“…Compared to nasopharyngeal swabs, saliva samples harbor similar levels of viral load while being easier to obtain via self-collection. Several groups have developed RT-LAMP tests to detect SARS-CoV-2 in saliva samples ( Bhadra et al, 2020 ; Flynn et al, 2020 ; Lalli et al, 2020 ; Lamb et al, 2020 ; Nagura-Ikeda et al, 2020 ; Rabe and Cepko, 2020 ; Taki et al, 2021 ; Yokota et al, 2020 ). However, due to pH variability between saliva samples, RT-LAMP often has a high rate of false positives when used with the common pH-dependent dye phenol red ( Bhadra et al, 2020 ; Hardinge and Murray, 2019 ).…”

An internet of things-based point-of-care device for direct reverse-transcription-loop mediated isothermal amplification to identify SARS-CoV-2

“…Although our LOD value was relatively higher than that of the conventional method, it was quite comparable to those produced by other POC systems with a direct RT-LAMP reaction ( appsec1 Table S2 ). Considering that exponential growth of virus occurs within 1-2 days after viral infection during which the viral load rise to 1000-fold higher than the LOD of qPCR (10 5 – 10 7 copies/mL) ( Flynn et al, 2020 ; Larremore et al, 2020 ), our proposed system can detect virus spikes by performing the COVID-19 surveillance testing.

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