Development of Human Rhinovirus RNA Reference Material Using Digital PCR

Abstract

The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV enhances its replication by concentrating RNA synthesis within a modified host membrane in an intracellular compartment. RV infections often occur alongside infections caused by other respiratory viruses, and the RV virus may remain asymptomatic for extended periods. Alongside qualitative detection, it is essential to accurately quantify RV RNA from clinical samples to explore the relationships between RV viral load, infections caused by the virus, and the resulting symptoms observed in patients. A reference material (RM) is required for quality evaluation, the performance evaluation of molecular diagnostic products, and evaluation of antiviral agents in the laboratory. The preparation process for the RM involves creating an RV RNA mixture by combining RV viral RNA with RNA storage solution and matrix. The resulting RV RNA mixture is scaled up to a volume of 25 mL, then dispensed at 100 µL per vial and stored at −80 °C. The process of measuring the stability and homogeneity of RV RMs was conducted by employing reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Digital PCR is useful for the analysis of standards and can help to improve measurement compatibility: it represents the equivalence of a series of outcomes for reference materials and samples being analyzed when a few measurement procedures are employed, enabling objective comparisons between quantitative findings obtained through various experiments. The number of copies value represents a measured result of approximately 1.6 × 105 copies/μL. The RM has about an 11% bottle-to-bottle homogeneity and shows stable results for 1 week at temperatures of 4 °C and −20 °C and for 12 months at a temperature of −80 °C. The developed RM can enhance the dependability of RV molecular tests by providing a precise reference value for the absolute copy number of a viral target gene. Additionally, it can serve as a reference for diverse studies.