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

Nucleic acid testing is a powerful tool for the detection of various pathogens. Respiratory syncytial virus (RSV) is a major cause of acute respiratory infection, especially in young children and infants. To improve the confidence and reliability of nucleic acid testing results for RSV, reference materials (RMs) of both type A and B of RSV were developed by the National Institute of Metrology, China, code numbers NIM-RM 4057 and 4058. The reference material was composed of in vitro transcribed RNA containing the nucleocapsid (N) gene, matrix (M) gene, and partial polymerase (L) gene of RSV. A duplex reverse transcription digital PCR method was established with limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) of 2, 5, and 23 copies per reaction for RSV-A and 4, 8, and 20 copies per reaction for RSV-B. The certified value and expanded uncertainty (U, k = 2) of the two RMs were determined to be (6.1 +/- 1.4) x-10(4) copies/mu L for RSV-A and (5.3 +/- 1.2) x-10(4) copies/mu L for RSV-B. The developed RMs can be used as standards to evaluate the performance of RSV detection assays.

Automated summary

Nucleic acid testing is an effective method for detecting pathogens, and reference materials for respiratory syncytial virus (RSV) have been developed to improve the reliability of RSV testing. These reference materials contain RNA with specific genes of RSV and a duplex reverse transcription digital PCR method was established with specific detection limits for RSV-A and RSV-B. The certified values and uncertainties of the reference materials were determined, and they can be used to evaluate the performance of RSV detection assays.