Simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex PCR - PubMed (original) (raw)

Simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex PCR

J E Echevarría et al. J Clin Microbiol. 1998 May.

Abstract

Reverse transcription (RT)-PCR assays have been widely described for use in the diagnosis of human parainfluenza viruses (HPIVs) and other respiratory virus pathogens. However, these assays are mostly monospecific, requiring separate amplifications for each HPIV type. In the present work, we describe multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction. Specifically, a mixture of three pairs of primers to conserved regions of the hemagglutinin-neuraminidase gene of each HPIV serotype was used for primary amplification, yielding amplicons with similar sizes. For typing, a second amplification was performed with a mixture of nested primers, yielding amplicons with sizes easily differentiated by agarose gel electrophoresis. A modified single-amplification RT-PCR assay with fluorescence-labeled nested primers, followed by analysis of the labeled products on an automated sequencing gel, was also evaluated. Fifteen temporally and geographically diverse HPIV isolates from the Centers for Disease Control and Prevention archives and 26 of 30 (87%) previously positive nasopharyngeal specimens (8 of 10 positive for HPIV serotype 1 [HPIV1], 9 of 10 positive for HPIV2, and 9 of 10 positive for HPIV3) were positive and were correctly typed by both assays. Negative results were obtained with naso- or oropharyngeal specimens and/or culture isolates of 33 unrelated respiratory tract pathogens, including HPIV4, enterovirus, rhinovirus, respiratory syncytial virus, adenovirus, influenza virus, and Streptococcus pneumoniae. Our multiplex RT-PCR assays provide sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.

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Figures

FIG. 1

FIG. 1

Detection and typing of prototype HPIV1, HPIV2, and HPIV3 strains. (A) Lane M, molecular weight markers; lanes 1, 2, and 3, HPIV1, HPIV2, and HPIV3, respectively; lane A, mixture of all three HPIV types. (B) GeneScan Analysis Software profile of the mixture in lane A.

FIG. 2

FIG. 2

Results of nested RT-PCR with clinical samples. No discrepancies with the results obtained by the method with GeneScan Analysis Software were found (data not shown).

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