Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network
| Autor: | Shailesh D. Pawar, Deepa Sharma, Everardo Vega, Ma. Anne-Lesley D. Valencia, Mehar Angez, Richter Razafindratsimandresy, Seta Andriamamonjy, Mark Mandelbaum, Uma P. Nalavade, Stacey Jeffries-Miles, Chelsea Harrington, Elisabeth Pukuta Simbu, Nancy Gerloff, S. Shahid Shaukat, Hong Sun, Lea Necitas G. Apostol |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
RNA viruses
Background fluorescence Viral Diseases Research Facilities Computer science Artificial Gene Amplification and Extension Pathology and Laboratory Medicine medicine.disease_cause Polymerase Chain Reaction Enteroviruses Geographical Locations Feces Medical Conditions Medicine and Health Sciences False positive paradox Multidisciplinary Sewage Poliovirus WHO method Infectious Diseases Real-time polymerase chain reaction Medical Microbiology Viral Pathogens Viruses Medicine Biological Cultures Pathogens Research Laboratories Research Article Asia Virus isolation Science Nucleotide sequencing Computational biology Real-Time Polymerase Chain Reaction Research and Analysis Methods Microbiology medicine Molecular Biology Techniques Microbial Pathogens Molecular Biology Biology and life sciences Organisms Reverse Transcriptase-Polymerase Chain Reaction Cell Cultures People and Places Laboratories Poliomyelitis Government Laboratories |
| Zdroj: | PLoS ONE, Vol 16, Iss 8, p e0255795 (2021) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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