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. 2009 Jun;47(6):1939-41.
doi: 10.1128/JCM.00702-09. Epub 2009 Apr 22.

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues

David R Kilpatrick  1 , Chen-Fu Yang, Karen Ching, Annelet Vincent, Jane Iber, Ray Campagnoli, Mark Mandelbaum, Lina De, Su-Ju Yang, Allan Nix, Olen M Kew
Affiliations

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues

David R Kilpatrick et al. J Clin Microbiol. 2009 Jun.

Abstract

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

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Figures

FIG. 1.
FIG. 1.
Specificities of poliovirus TaqMan rRT-PCR primers and probes, demonstrated by using Sabin strain RNA templates (S1, Sabin 1; S2, Sabin 2; S3, Sabin 3; NTC, no-template control). Each assay (and fluorophore) was performed singly to demonstrate individual assay specificities: (A) panEV (FAM); (B) panPV (FAM); (C) seroPV1 (FAM); (D) seroPV2 (FAM); (E) seroPV3 (FAM); (F) Sab1 assay (CY5); (G) Sab2 assay (FAM); and (H) Sab3 assay (ROX). The no-template control was used to manually set the zero baseline fluorescence emissions. The efficiency of each assay was >90%, based on 10-fold dilutions of control RNAs (10 ng to 1 pg). Cycle threshold values of 30 or more were observed to approach the sensitivity limits of the real-time detection system; therefore, cycle threshold values of <30 were considered positive detections of the target template.
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