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Journal of Clinical Microbiology, August 2006, p. 2698-2704, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.00542-06

Sensitive, Seminested PCR Amplification of VP1 Sequences for Direct Identification of All Enterovirus Serotypes from Original Clinical Specimens

W. Allan Nix, M. Steven Oberste,* and Mark A. Pallansch

Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 13 March 2006/ Returned for modification 24 April 2006/ Accepted 24 May 2006

A reverse transcription-seminested PCR (RT-snPCR) assay was developed for the detection and identification of enterovirus (EV) RNA in clinical specimens. Three conserved protein motifs were identified by aligning the VP3 and VP1 sequences of prototype EV strains. Consensus degenerate primers were designed from a conserved VP3 motif and a distal VP1 motif for the first PCR. Consensus-degenerate hybrid oligonucleotide primers were designed from an internal VP1 motif and used with the same distal VP1 motif for the second, seminested PCR step. The primers were designed for broad target specificity and amplified all recognized and proposed EV serotypes and other antigenic variant strains tested. The VP1 RT-snPCR assay was slightly more sensitive than our in-house EV 5' nontranslated region RT-snPCR assay, detecting as few as 10 RNA copies per reaction. As an example application, the VP1 RT-snPCR assay was used to identify EVs in clinical specimens. A product of the expected size was successfully amplified and sequenced from cerebrospinal fluid; serum; stool suspensions; and nasopharyngeal, eye, and rectal swab specimens, allowing unambiguous identification of the infecting virus in all cases. The VP1 sequences derived from the RT-snPCR products allow rapid phylogenetic and molecular epidemiologic analysis of strains circulating during the EV season and comparison with EV sequences from past seasons or from different locations around the world.

* Corresponding author. Mailing address: Enterovirus Section, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333. Phone: (404) 639-5497. Fax: (404) 639-4011. E-mail: soberste{at}cdc.gov.

Journal of Clinical Microbiology, August 2006, p. 2698-2704, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.00542-06




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