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Journal of Clinical Microbiology, May 1998, p. 1388-1391, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Simultaneous Detection and Identification of Human Parainfluenza Viruses 1, 2, and 3 from Clinical Samples by Multiplex PCR

Juan E. Echevarría,1,2,* Dean D. Erdman,1 Ella M. Swierkosz,3 Brian P. Holloway,4 and Larry J. Anderson1

Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases,1 and DNA Section, Scientific Resources Program,4 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333; Servicio de Microbiología Diagnóstica, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo s/n, 28220 Majadahonda, Madrid, Spain2; and Department of Pathology and Pediatrics, St. Louis University, St. Louis, Missouri 631103

Received 9 October 1997/Returned for modification 17 December 1997/Accepted 13 February 1998

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.

* Corresponding author. Mailing address: Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo s/n, 28220 Majadahonda, Madrid, Spain. Phone: 34-1-5097901. Fax: 34-1-5097966. E-mail: jeecheva{at}isciii.es.

Journal of Clinical Microbiology, May 1998, p. 1388-1391, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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