Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control

Area of Science:

• Virology
• Molecular Biology
• Clinical Diagnostics

Background:

• Accurate measurement of SARS-CoV-2 viral load is crucial for managing COVID-19 patients and carriers.
• Clinical settings require reliable methods for SARS-CoV-2 quantification to inform patient care and public health strategies.

Purpose of the Study:

• To establish a feasible clinical assay for quantifying SARS-CoV-2 viral copy number.
• To implement an internal control for assessing sample quality and assay reliability.

Main Methods:

• Development of a duplex RT-qPCR assay targeting the SARS-CoV-2 E gene and the human RNase P gene.
• Utilizing RNase P amplification as an internal control for sample sufficiency and integrity.
• Establishing a standard curve for converting Cq values to viral copy numbers.

Main Results:

• The duplex RT-qPCR assay is clinically feasible and highly reproducible, linear across 7 logs.
• An internal control identified 2.4% of nasopharyngeal swabs as inadequate for testing.
• SARS-CoV-2 copy number was independent of RNase P copy number, ensuring comparability between samples.

Conclusions:

• Quantification of SARS-CoV-2 viral load is achievable in a clinical setting.
• The assay's internal control is vital for detecting inadequate samples and preventing false negatives.
• Accurate viral load quantification facilitates evidence-based clinical and public health decision-making for COVID-19.