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Updated: Oct 25, 2025
Particle Agglutination Method for Poliovirus Identification
Published on: April 20, 2011
Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory
Hong Sun1, Chelsea Harrington1, Nancy Gerloff1
1Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
Abstract:
Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.
Insights
A new poliovirus probe improves real-time RT-PCR assays for global polio surveillance. This enhanced assay reduces background noise and false positives, aiding in poliovirus detection and eradication efforts.
Area of Science:
- Virology
- Molecular Biology
- Public Health
Background:
- Poliovirus (PV) surveillance is critical for global eradication efforts.
- Intratypic differentiation (ITD) using real-time RT-PCR is a key step in PV surveillance.
- Validation of real-time PCR platforms is essential for accurate PV detection.
Purpose of the Study:
- To validate the performance of four real-time PCR platforms using an ITD kit.
- To evaluate a redesigned poliovirus probe with a double-quencher (Zen™) for improved Pan-poliovirus (PanPV) assay performance.
- To assess the utility of the updated PanPV probe in global polio laboratories.
Main Methods:
- Validation of four real-time PCR platforms (ABI7500, Bio-Rad CFX96, Stratagene MX3000P, Qiagen Rotor-Gene Q) with the ITD kit.
- Re-design of the poliovirus probe in the PanPV assay with a double-quencher to reduce background fluorescence.
- Evaluation of the updated PanPV probe using a panel of 184 polioviruses and non-polio enteroviruses.
- Pilot testing of the new assay in five Global Polio Laboratory Network (GPLN) laboratories.
Main Results:
- The redesigned poliovirus probe demonstrated reduced background fluorescence.
- The updated PanPV probe decreased the number of false positives compared to the standard probe.
- The assay was successfully pilot tested in diverse international laboratory settings.
Conclusions:
- The redesigned double-quencher poliovirus probe enhances the performance of real-time RT-PCR assays for poliovirus detection.
- This improvement is crucial for accurate and reliable poliovirus surveillance and monitoring eradication progress.
- The updated assay simplifies interpretation and ensures capability across various real-time PCR platforms.
