Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Development

Background:

• Widespread diagnostic testing is crucial for controlling COVID-19 transmission.
• Current RT-PCR tests require RNA purification, adding complexity and time.
• Sensitive detection of SARS-CoV-2, including in asymptomatic cases, is vital.

Purpose of the Study:

• To develop a simplified and sensitive method for SARS-CoV-2 RNA detection.
• To enable direct RT-qPCR without RNA purification using a novel processing technique.

Main Methods:

• Developed Alkaline-Glycol (AG) Processing: biological specimens treated at pH 12.2-12.8 for 5 minutes at room temperature.
• Direct RT-qPCR performed on AG-processed specimens (saliva or swab suspensions).
• Evaluated sensitivity and compatibility with various RT-qPCR kits and specimen types.

Main Results:

• AG Processing effectively lyses viruses and reduces RT-PCR inhibitors.
• Sensitivity of viral RNA detection is comparable to methods with RNA purification.
• Limit of Detection (LOD) of 300 viral copies/mL saliva achieved.
• Method compatible with saliva and swab specimens in Universal Transport Medium.

Conclusions:

• AG Processing offers a simplified, sensitive, and reliable direct RT-qPCR method for COVID-19 diagnostics.
• The technique is scalable and adaptable for automated workflows.
• This method supports effective mass screening and pandemic management.