Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network.

Autor: Sun H; Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America., Harrington C; Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America., Gerloff N; Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America., Mandelbaum M; Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America., Jeffries-Miles S; Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America., Apostol LNG; Research Institute for Tropical Medicine, Muntinlupa City, Philippines., Valencia MAD; Research Institute for Tropical Medicine, Muntinlupa City, Philippines., Shaukat S; National Institute of Health, Islamabad, Pakistan., Angez M; National Institute of Health, Islamabad, Pakistan., Sharma DK; National Institute of Virology, Mumbai, India., Nalavade UP; National Institute of Virology, Mumbai, India., Pawar SD; National Institute of Virology, Mumbai, India., Pukuta Simbu E; National Institute of Biomedical Research, Kinshasa, Democratic Republic of the Congo, Congo., Andriamamonjy S; Virology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar., Razafindratsimandresy R; Virology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar., Vega E; Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2021 Aug 06; Vol. 16 (8), pp. e0255795. Date of Electronic Publication: 2021 Aug 06 (Print Publication: 2021).
DOI: 10.1371/journal.pone.0255795
Abstrakt: 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.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
Externí odkaz: