Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR

Abstract

Timely diagnosis of respiratory syncytial virus (RSV) infection is critical for appropriate treatment of lower respiratory infection in young children. To facilitate diagnosis, we developed a rapid, specific, and sensitive TaqMan PCR method for detection of RSV A and RSV B. Two sets of primer-probe pairs were selected from the nucleotide sequences encoding the nucleocapsid protein--one targeting RSV A and the other targeting RSV B. The specificity of the TaqMan reverse transcription-PCR assay was evaluated by testing each primer-probe pair against various viruses derived from laboratory virus stocks, as well as clinical respiratory specimens. Fluorescent signals were observed only in the presence of RSV A and/or RSV B. The sensitivity of our quantitative PCR assay was determined on the basis of PFU and virus particle counts. The resulting assay sensitivity was found to be 0.023 PFU, or two copies of viral RNA, for RSV A and 0.018 PFU, or nine copies of viral RNA, for RSV B. This quantitative TaqMan PCR assay was utilized to diagnose 175 nasopharyngeal aspirates obtained from children in Hong Kong with respiratory symptoms during the winter of 2000 and 2001. Among these specimens, TaqMan PCR detected 36 RSV-positive samples, 10 of which were identified as RSV A and 26 of which were identified as RSV B, whereas culture confirmation identified 21 RSV-positive specimens and immunofluorescence identified 32 RSV-positive specimens, all of which were among those identified by PCR. The results confirmed the accuracy of our TaqMan PCR assay and demonstrated its improved sensitivity versus classical methods.

FIG. 1.

Specificity of RSV primer-probe pairs. RSV A primer pair A21-A102 and probe APB48 (a) and RSV B primer pair B17-B120 and probe BPB45 (b) were employed to detect RSV A2 (▪) and 2B (⧫) RNAs.

FIG. 2.

Sensitivity of the TaqMan RT-PCR assay. Tenfold serial dilutions of the RNA extracted from RSV A2 (0.0023 PFU to 230 PFU) (a) and RSV 2B (0.0018 PFU to 180 PFU) (b) were used in the PCR. Primer-probe pairs specific for RSV A (a) and B (b) were employed to detect the corresponding targets. The negative control contained RNase-free water instead of RNA template.

FIG. 2.

Sensitivity of the TaqMan RT-PCR assay. Tenfold serial dilutions of the RNA extracted from RSV A2 (0.0023 PFU to 230 PFU) (a) and RSV 2B (0.0018 PFU to 180 PFU) (b) were used in the PCR. Primer-probe pairs specific for RSV A (a) and B (b) were employed to detect the corresponding targets. The negative control contained RNase-free water instead of RNA template.

FIG. 3.

RSV-positive clinical specimens analyzed by TaqMan PCR. The samples were collected and analyzed as described in Materials and Methods. The threshold cycle (C_T) represents the cycle number at which a significant increment of fluorescence signal is first detected. Each symbol represents the C_T number for an individual specimen.