Novel COVID-19 test delivers quick, accurate, cost-efficient virus detection

Researchers have recently developed a novel reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) test that can rapidly detect the presence SARS-CoV-2 in clinical samples with high accuracy and without the need for RNA extraction.

“With high sensitivity, specificity, and bypassing the need for RNA extraction, the RT-LAMP Rapid Detection assay is a valuable and fast test for an accurate and rapid RNA detection of the SARS-CoV-2 virus and potentially other pathogens,” the researchers said.

“Additionally, the versatility of this test allows its application in virtually every laboratory setting and remote location where access to expensive laboratory equipment is a limiting factor for testing during pandemic crises,” they added.

Sensitivity analysis revealed that the RT-LAMP test’s limit of detection was 80 copies/µL, at which concentration the test could detect the presence of the intact virus with 100 percent reliability and reproducibility. At 70 copies/µL, the rapid detection test could only identify 91.7 percent of samples with SARS-CoV-2. [PLoS One 2022;doi:10.1371/journal.pone.0266703]

Moreover, the primers used in the assay were found to be a 100-percent match against the reference genome sequences of SARS-CoV-2, pointing to the high specificity of the RT-LAMP test.

Subsequent mapping against the sequences in the Global Initiative on Sharing Avian Influenza Data database found that poor hybridization of all three primer sets used in the novel test would be exceedingly rare, occurring at a rate of around 1 in 140,810 instances.

The researchers then tested the RT-LAMP assay on nasopharyngeal swabs, obtained from patients confirmed to be positive (symptomatic and asymptomatic) and negative for SARS-CoV-2. When compared against an FDA-approved test, RT-LAMP achieved a 100-percent detection rate.

“Although the assay represents a valuable tool for the SARS-CoV-2 detection in clinical samples, significant limitations must be considered,” the researchers said. These include the possibility of mutations in LAMP’s target genomic regions, which could eventually lead to false negatives, and the lack of accounting for emerging vaccines and therapeutics for the coronavirus disease 2019 (COVID-19).

Moreover, negative results do not necessarily mean true negatives, the researchers added, cautioning that novel RT-LAMP “should not be used as the sole basis for treatment.”

Accurate, fast, economical

Currently, the most accurate tests for COVID-19 are labour- and resource-intensive, requiring lengthy RNA extraction and quality assessment steps, as well as expensive machines and reagents for reverse-transcription polymerase chain reaction (PCR). Meanwhile, rapid assays suffer from low accuracies and high false-positivity rates.

The novel RT-LAMP assay, in contrast, attempts to solve both problems at once. “Because the method is based on an isothermal step, it does not require expensive PCR equipment. It could be quickly executed on a regular thermal cycler combined with a plate reader,” the researchers said.

However, its strongest advantage is perhaps that no RNA extraction is needed. After collection, samples are mixed in with a buffer that keeps viral RNA stable at room temperature. The test also requires no preincubation or enzyme activation step, leading to the overall effect of shorter turnaround times.

“Taken together, our data propose an entire pipeline (from sample collection to data visualization) that can be efficiently executed in less than 1 hour and presents a high level of versatility and adaptability not only to laboratory settings but also to impromptu testing sites,” the researchers said.