A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed...

Area of Science:

• Molecular Biology
• Virology
• Public Health

Background:

• The gold standard for diagnosing coronavirus disease 2019 (COVID-19) involves RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes.
• This established method, while accurate, is costly and uses invasive nasopharyngeal swabs, limiting its utility for widespread public health screening.
• There is a need for more accessible, cost-effective, and less invasive diagnostic tools for large-scale infectious disease surveillance.

Purpose of the Study:

• To develop and validate a novel, one-step RT-PCR test utilizing the DNA-intercalating dye SYBR Green for detecting SARS-CoV-2.
• To assess the feasibility of using saliva as a sample type for this RT-PCR assay, aiming for a less invasive collection method.
• To evaluate the test's ability to identify SARS-CoV-2 variants of concern and its performance in a real-world mass-screening setting.

Main Methods:

• A one-step RT-PCR assay was developed using SYBR Green dye for direct detection of SARS-CoV-2 RNA from saliva or nasopharyngeal (NP) swabs.
• The assay's sensitivity, specificity, and ability to discriminate between SARS-CoV-2 variants were evaluated.
• The developed saliva RT-PCR SYBR Green test was implemented in a mass-screening initiative involving approximately 4500 asymptomatic children.

Main Results:

• The SYBR Green-based RT-PCR test demonstrated reliable detection of SARS-CoV-2 from both saliva and NP swabs, comparable to the gold standard.
• The assay could be optimized to differentiate between SARS-CoV-2 variants of concern.
• In a mass-screening of children, the saliva RT-PCR test proved effective and cost-efficient, outperforming NP rapid antigen tests in some cases.

Conclusions:

• RT-PCR saliva tests employing DNA-intercalating dyes like SYBR Green offer a powerful, reliable, and cost-effective strategy for community screening of SARS-CoV-2.
• This approach provides a less invasive alternative to traditional NP swab testing, enhancing compliance and feasibility for mass screening.
• The methodology is adaptable for detecting other infectious agents, positioning it as a valuable tool for future pandemic preparedness and response.