GeneScan reverse transcription-PCR assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness

Abstract

A reverse transcription-PCR (RT-PCR) assay based on automated fluorescent capillary electrophoresis and GeneScan software analysis was developed to detect six common respiratory viruses in clinical specimens from young children. Assays for human respiratory syncytial virus (HRSV); human parainfluenza viruses 1, 2, and 3 (HPIV1, -2, and -3, respectively); and influenza A and B viruses were incorporated into a single standard assay format. The optimized assay panel was used to test 470 respiratory specimens obtained from 462 children hospitalized with acute respiratory illness that had been previously tested by viral culture (405 specimens) or direct immunofluorescence staining (DIF) (65 specimens). Of 93 specimens positive for respiratory viruses by culture or DIF, 86 (92%) were positive by RT-PCR, including 66 HRSV, 2 HPIV2, 5 HPIV3, 3 influenza A virus, and 10 influenza B virus specimens. An additional 119 respiratory viruses were identified by RT-PCR in 116 patients for whom results were negative by viral isolation or DIF. We conclude that the GeneScan RT-PCR panel can markedly improve detection of acute respiratory virus infections in young children.

FIG. 1.

Electropherogram of GeneScan analysis results showing RT-PCR amplification products for respiratory virus and β-actin controls run under denaturing conditions.

FIG. 2.

Comparison of GeneScan (A) with ethidium bromide staining (B) for analysis of RT-PCR products obtained from serial fivefold dilutions of a plasmid containing cloned HRSV fragment. The mean product sizes (nucleotides) obtained by GeneScan from duplicate samples for each dilution are indicated under the data points. Arrows indicate detection limits for each method.