Direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-PCR assay

Abstract

Diagnosis of respiratory virus infections currently involves detection by isolation or antigen detection, which usually identifies only a single suspected agent. To permit identification of more than one respiratory virus in clinical specimens, a rapid detection method involving a single-step, multiplex reverse transcription-PCR (RT-PCR) assay was developed. The assay included five primer sets that amplified the RNA of respiratory syncytial virus subtypes A and B, parainfluenza virus types 1, 2, and 3, and adenovirus types 1 to 7. Initially the assay was tested on tissue culture-grown virus and was found to be specific for all 12 prototype viruses tested, with no interassay cross amplification or amplification of other respiratory viruses. Assay sensitivity allowed a detection range of 0.2 50% tissue culture infectious dose (TCID50) for adenovirus to 250 TCID50 for parainfluenza virus type 1. The multiplex RT-PCR assay was also able to directly detect viruses in respiratory specimens, with virus being detected in 41 of 112 samples as compared to 34 of 112 samples detected by direct immunofluorescence or antigen detection following specimen culture. This suggests that the multiplex RT-PCR assay can be used as a rapid and sensitive diagnostic method for major respiratory viruses.

FIG. 1

Multiplex RT-PCR detection of RSV-A and RSV-B. (A) RNA extracted from RSV-A- or RSV-B-infected HEp-2 cells was reverse transcribed and amplified under the conditions described in Materials and Methods. PCR products were separated on a 1% agarose gel which was stained with ethidium bromide and visualized under UV light. Lanes: 1, fluorescein-11-dUTP-labeled base pair marker; 2, RSV-A; 3, RSV-B. (B) Southern hybridization of the same gel in panel A hybridized to RSV subtype-specific oligonucleotide probes 3′ end labeled with fluorescein-11-dUTP. The blot was stripped and reprobed with the second probe according to procedures described in Materials and Methods. Blot 1, probed with rsva; blot 2, probed with rsvb.

FIG. 2

Single-tube, multiplex RT-PCR detection of five respiratory virus types. RNA extracted from RSV-A-, PIV3-, adenovirus type 5-, PIV2-, and PIV1-infected cells was pooled and reverse transcribed and amplified in a single tube under conditions described in Materials and Methods. RNA extracted from uninfected HEp-2 and LLC-MK2 cells was pooled and used as a negative control. PCR products were separated on a 3% NuSieve agarose gel that was stained with ethidium bromide and visualized under UV light. Lanes: 1, Template-free negative control; 2, pooled, uninfected-cell RNA; 3, pooled, infected-cell RNA; 4, 100-bp ladder DNA marker (Boehringer Mannheim). Expected sizes for the five virus type amplicons are as follows: for RSV, 348 bp; for PIV3, 234 bp; for adenovirus, 215 bp; for PIV2, 164 bp; and for PIV1, 84 bp.

FIG. 3

Multiplex RT-PCR detection of respiratory viruses in respiratory specimens. (A) Respiratory specimens were processed for detection of viral RNA by multiplex RT-PCR as described in Materials and Methods. PCR products were separated on a 1% agarose gel which was stained with ethidium bromide and visualized under UV light. The results from a representative group of respiratory specimens are shown. The antigen detection result and, in parentheses, the RT-PCR result for each specimen were as follows: lane 1, virus negative (virus negative); lane 2, RSV (RSV); lane 3, virus negative (adenovirus); lane 4, RSV (RSV); lane 5, virus negative (virus negative); and lane 6, PIV3 (PIV3 and adenovirus). (B) Southern hybridization of the same gel in panel A hybridized to oligonucleotide probes 3′ end labeled with fluorescein-11-dUTP. The blot was stripped and reprobed with each individual probe according to procedures described in Materials and Methods. The sizes of a fluorescein-labeled DNA marker (Lambda DNA digested with EcoT14I; Amersham Life Science) run on the transferred gel are shown to the right of each reprobed blot. (C) Gel (3% NuSieve agarose) of multiplex RT-PCR products from the PIV3 antigen-positive respiratory specimen shown in lane 6 of panel A. Lanes: 1, 100-bp ladder DNA marker; 2, respiratory specimen positive for PIV3 by antigen detection (PIV3-specific amplicon band, 234 bp; adenovirus-specific amplicon band, 215 bp).