Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Clinical Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Virology

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

W. Allan Nix, M. Steven Oberste, Mark A. Pallansch
W. Allan Nix
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
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
M. Steven Oberste
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
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: soberste@cdc.gov
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
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JCM.00542-06
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • FIG. 1.
    • Open in new tab
    • Download powerpoint
    FIG. 1.

    Schematic representation of the locations of the primers used in the CODEHOP VP1 RT-snPCR. (A) Similarity plot of the aligned capsid amino acid sequences of 64 enterovirus prototype strains. Sequence identity scores were calculated within each 6-residue window, and the window was progressively moved across the alignment in 1-residue increments, with the identity score plotted versus the position at the center of the window. The positions of the four mature EV capsid proteins, VP4, VP2, VP3, and VP1, are shown at the top. The orientations and approximate positions of the cDNA primers (open arrowheads) and PCR primers (filled arrowheads) are shown directly above the plot. (B) Amino acid motifs used in primer design and schematic representation of the steps in the CODEHOP VP1 RT-snPCR assay. Consensus amino acid motifs are shown. Asterisks indicate that the residue directly above the asterisk is present at that position in at least 90% of EV prototype strains; when only a single residue is shown, it is present in all prototype strains. Primer sequences are shown directly below the amino acid motif sequences. Ambiguity codes are as follows: R, A or G; Y, C or T; W, A or T; N, A, C, G, or T; I, inosine.

  • FIG. 2.
    • Open in new tab
    • Download powerpoint
    FIG. 2.

    Sensitivity of VP1 RT-snPCR and comparison of the sensitivity with that of the 5′ nontranslated region RT-snPCR. (A) Amplification of RNA extracted from 10-fold serial dilutions of an EV68 virus stock; (B) amplification of 10-fold serial dilutions of VP3-VP1 sRNA; (C) comparison of VP1 RT-snPCR (top) with 5′-NTR RT-snPCR (bottom) using 10-fold serial dilutions.

  • FIG. 3.
    • Open in new tab
    • Download powerpoint
    FIG. 3.

    VP1 RT-snPCR amplification of RNA extracted directly from original clinical specimens. For each reaction, 50 μl of each seminested PCR2 product was analyzed and gel purified by electrophoresis on a 1.5% agarose gel containing 0.5 μg ethidium bromide per milliliter. The specimens tested were cerebrospinal fluid (CSF), stool, rectal swab (RS), nasopharyngeal swab (NPS), eye (conjunctival) swab (ES), serum, and postmortem liver tissue.

Tables

  • Figures
  • TABLE 1.

    Primers used for cDNA synthesis, PCR amplification, and sequencing

    PrimerSequenceAmino acid motifGeneLocationa
    AN32GTYTGCCAWQTVP13009-3002
    AN33GAYTGCCAWQSVP13009-3002
    AN34CCRTCRTAYDGVP13111-3104
    AN35RCTYTGCCAWQSVP13009-3002
    224GCIATGYTIGGIACICAYRTAMLGTH(I/L/M)VP31977-1996
    222CICCIGGIGGIAYRWACATM(F/Y)(I/V)PPG(A/G)VP12969-2951
    292MIGCIGYIGARACNGG(Q/T)A(A/V)ETGVP12612-2627
    AN89 CCAGCACTGACAGCAGYNGARAYNGGbPALTA(A/V)E(I/T)GVP12602-2627
    AN88 TACTGGACCACCTGGNGGNAYRWACATbM(F/Y)(I/V)PPGGPVVP12977-2951
    AN232CCAGCACTGACAGCAbPALTAVP12602-2616
    AN233TACTGGACCACCTGGbPGGPVVP12977-2963
    AN230 AATTAACCCTCACTAAAGGGAGA AGATATTATACTCAYTGGcRYYTHWVP31993-2010
    AN231GTCAGCTGGGTTTATNCCRTAYGINPADVP13069-3049
    • ↵ a The locations of all primers except AN230 and AN231 are those relative to the genome of PV1 Mahoney (GenBank accession number J02281 ); the locations for AN230 and AN231 are relative to those of the genome of EV68 Fermon (GenBank accession number AY426531 ).

    • ↵ b AN232 is the nondegenerate “clamp” portion of AN89, and AN233 is the nondegenerate clamp portion of AN88. Within the AN88 and AN89 sequences, these clamp regions are indicated in italic type.

    • ↵ c The T3 RNA polymerase promoter sequence is underlined.

  • TABLE 2.

    Enterovirus reference strains amplified by VP1 RT-snPCRa

    SerotypeStrainSerotypeStrain
    CVA1TompkinsE16Harrington
    CVA2FleetwoodE17CHHE-29
    CVA3OlsonE18Metcalf
    CVA4High PointE19Burke
    CVA5SwartzE20JV-1
    CVA6GdulaE21Farina
    CVA7AB-IVE24De Camp
    CVA8DonovanE25JV-4
    CVA9GriggsE26Coronel
    CVA10KowalikE27Bacon
    CVA11Belgium-1E29JV-10
    CVA12Texas-12E30Bastianni
    CVA13Flores30Frater
    CVA14G-14E30Giles
    CVA15G-9E30PR-17
    CVA16G-10E31Caldwell
    CVA17G-12E32PR-10
    CVA18G-13E33Toluca-3
    CVA198663EV68Fermon
    CVA20IH-35EV69Toluca-1
    CVA21KuykendallEV70J670/71
    CVA22ChulmanEV71BrCr
    CVA24JosephEV73CA55-1988
    CVA24EH24EV7410213
    CVA24DN-19 (E34)EV7510362
    CVB1Conn-5EV7610369
    CVB2Ohio-1EV7910384
    CVB3NancyEV8010387
    CVB4JVBEV8110389
    CVB5FaulknerEV8210390
    CVB6SchmittEV8310392
    E1FaroukEV8410603
    E1Bryson (E8)EV8510353
    E2CornelisEV8610354
    E3MorriseyEV8710396
    E4Du ToitEV8810398
    E4ShropshireEV8910395
    E4PesacekEV9010399
    E5NoyceEV9110406
    E6D;AmoriEV9210408
    E6CoxEV9610358
    E6BurgessEV9710355
    E6CharlesEV10010500
    E7WallaceEV10110361
    E9HillPV1Mahoney
    E11GregoryPV1Sabin
    E11SilvaPV2Lansing
    E12TravisPV2Sabin
    E13Del CarmenPV2Lansing
    E14TowPV3Leon
    E15CH96-51PV3Sabin
    • ↵ a Reference strains for EV77 to EV78 and EV93 to EV95 were not tested because they are not publicly available. Some other numbers are missing due to reclassification (i.e., coxsackievirus A serotype 23 [CVA23] is a variant of echovirus 9 [E9], E8 is a variant of E1, E34 is a variant of CVA24, E10 is reovirus 1 [genus Orthoreovirus, family Reoviridae], E28 is human rhinovirus 1A [genus Rhinovirus, family Picornaviridae], and EV72 is human hepatitis A virus [genus Hepatovirus, family Picornaviridae]). The “newer” serotypes (EV73 to EV76, EV79 to EV92, EV96, EV97, EV100, and EV101) were identified and classified by molecular means rather than by antigenic means (15, 18, 21; M. S. Oberste, unpublished data). PV, poliovirus.

  • TABLE 3.

    Clinical isolates amplified by VP1 RT-snPCR

    SerotypeNo. of isolatesYr(s) of isolation
    CVA1421992-1994
    CVA1641984-1995
    CVA2021983
    CVA2161986-1996
    CVA2411984
    CVA921993-1996
    CVB231982-1995
    CVB361988-1997
    CVB411984
    CVB541983-1993
    E331986-1988
    E421985-1988
    E511996
    E631991-1998
    E731993-1998
    E931992-1995
    E1151988-1998
    E1211988
    E1321986-1995
    E1861985-1997
    E2111994
    E2411983
    E2561984-1994
    E2911988
    E3041991-1995
    E3311998
    EV7171988-1995
    EV7541985-1987
    HRV211990
    HRV3111988
PreviousNext
Back to top
Download PDF
Citation Tools
Sensitive, Seminested PCR Amplification of VP1 Sequences for Direct Identification of All Enterovirus Serotypes from Original Clinical Specimens
W. Allan Nix, M. Steven Oberste, Mark A. Pallansch
Journal of Clinical Microbiology Aug 2006, 44 (8) 2698-2704; DOI: 10.1128/JCM.00542-06

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Clinical Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Sensitive, Seminested PCR Amplification of VP1 Sequences for Direct Identification of All Enterovirus Serotypes from Original Clinical Specimens
(Your Name) has forwarded a page to you from Journal of Clinical Microbiology
(Your Name) thought you would be interested in this article in Journal of Clinical Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Sensitive, Seminested PCR Amplification of VP1 Sequences for Direct Identification of All Enterovirus Serotypes from Original Clinical Specimens
W. Allan Nix, M. Steven Oberste, Mark A. Pallansch
Journal of Clinical Microbiology Aug 2006, 44 (8) 2698-2704; DOI: 10.1128/JCM.00542-06
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Capsid Proteins
enterovirus
Enterovirus Infections
Molecular Diagnostic Techniques
polymerase chain reaction

Related Articles

Cited By...

About

  • About JCM
  • Editor in Chief
  • Board of Editors
  • Editor Conflicts of Interest
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Resources for Clinical Microbiologists
  • Ethics
  • Contact Us

Follow #JClinMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

 

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0095-1137; Online ISSN: 1098-660X