Reference material development for detection of human respiratory syncytial virus using digital PCR

Area of Science:

• Molecular Biology
• Virology
• Metrology

Background:

• Nucleic acid testing is crucial for pathogen detection, with Respiratory Syncytial Virus (RSV) being a significant cause of acute respiratory infections, particularly in vulnerable populations.
• Ensuring the accuracy and reliability of RSV diagnostic tests is paramount for effective patient management and public health surveillance.

Purpose of the Study:

• To develop and characterize high-quality reference materials (RMs) for Respiratory Syncytial Virus (RSV) types A and B.
• To establish robust standards for validating and improving the performance of RSV nucleic acid detection assays.

Main Methods:

• In vitro transcribed RNA, containing key genes (N, M, partial L) of RSV types A and B, was used to create the reference materials.
• A duplex reverse transcription digital PCR (RT-dPCR) method was optimized to determine the analytical performance characteristics, including limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ).
• Certified values and expanded uncertainties for RSV-A and RSV-B concentrations were established using the developed RT-dPCR method.

Main Results:

• Reference materials NIM-RM 4057 (RSV-A) and NIM-RM 4058 (RSV-B) were successfully developed.
• The duplex RT-dPCR assay demonstrated sensitive detection with LoD values of 5 copies/reaction for RSV-A and 8 copies/reaction for RSV-B.
• Certified values were determined as (6.1 ± 1.4) × 10⁴ copies/μL for RSV-A and (5.3 ± 1.2) × 10⁴ copies/μL for RSV-B, with an expanded uncertainty (k=2).

Conclusions:

• The newly developed RSV-A and RSV-B reference materials provide reliable standards for the metrological traceability of quantitative nucleic acid testing.
• These RMs are essential tools for laboratories to assess the performance, ensure the quality, and enhance the confidence in RSV diagnostic assays.