Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests

Abstract

Diagnosis of any infectious disease is vital for opportune treatment and to prevent dissemination. RT-qPCR tests for detection of SARS-CoV-2, the causative agent for COVID-19, are ideal in a hospital environment. However, mass testing requires cheaper and simpler tests, especially in settings that lack sophisticated machinery. The most common current diagnostic method is based on nasopharyngeal sample collection, RNA extraction, and RT-qPCR for amplification and detection of viral nucleic acids. Here, we show that samples obtained from nasopharyngeal swabs in VTM and in saliva can be used with or without RNA purification in an isothermal loop-mediated amplification (LAMP)-based assay, with 60–93% sensitivity for SARS-CoV-2 detection as compared to standard RT-qPCR tests. A series of simple modifications to standard RT-LAMP published methods to stabilize pH fluctuations due to salivary acidity resulted in a significant improvement in reliability, opening new avenues for efficient, low-cost testing of COVID-19 infection.

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

“…Saliva is a complex biological mixture that can vary widely in pH, color, and viscosity. These factors can impair amplification efficiency and influence the performance of the test [ 36 ]. The characteristics that affect saliva viscosity include the presence of aggregates, variations in temperature, and the time elapsed between sample collection and testing.…”

A rapid, specific, extraction-less, and cost-effective RT-LAMP test for the detection of SARS-CoV-2 in clinical specimens

“…However, samples require refrigeration and must be analyzed within a short time frame. Additionally, RT-LAMP combined with a colorimetric read-out poses challenges for data interpretation due to high ambiguity and pH fluctuations can easily alter the readout [ 10 ].…”

Development and Implementation of a Simple and Rapid Extraction-Free Saliva SARS-CoV-2 RT-LAMP Workflow for Workplace Surveillance

“…Colorimetric LAMP uses the pH-sensitive dye phenol red which turns from pink/red to yellow at pH 6.8 and lower, following the generation of hydrogen ions resulting from amplification of the target [52][53][54]. Low pH saliva can cause reactions to instantaneously change to yellow pre-amplification and, if not noted, will result in a false positive determination [29,36]. In one study, 7% of saliva samples tested triggered a color change without amplification [11], and in another 15% of specimens showed a discordant color output when compared with an agarose gel electrophoresis readout [30].…”