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Optimizing PCR Detection of Zika Virus from Various Body Fluids

Current diagnostic protocols of acute Zika virus (ZIKV) infection focus on detection of viral RNA in serum or urine using reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, detecting infection can be a challenge, given that 80% of people with acute ZIKV infection are as...

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Autores principales: Gorchakov, Rodion, Berry, Rebecca M., Patel, Shital M., El Sahly, Hana M., Ronca, Shannon E., Murray, Kristy O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society of Tropical Medicine and Hygiene 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367632/
https://www.ncbi.nlm.nih.gov/pubmed/30560770
http://dx.doi.org/10.4269/ajtmh.18-0755
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author Gorchakov, Rodion
Berry, Rebecca M.
Patel, Shital M.
El Sahly, Hana M.
Ronca, Shannon E.
Murray, Kristy O.
author_facet Gorchakov, Rodion
Berry, Rebecca M.
Patel, Shital M.
El Sahly, Hana M.
Ronca, Shannon E.
Murray, Kristy O.
author_sort Gorchakov, Rodion
collection PubMed
description Current diagnostic protocols of acute Zika virus (ZIKV) infection focus on detection of viral RNA in serum or urine using reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, detecting infection can be a challenge, given that 80% of people with acute ZIKV infection are asymptomatic, and the window to detect viremia in serum is short. The ability to extend that window is needed to detect ZIKV at later time points after infection, particularly in high-risk individuals such as pregnant women. We evaluated RNA extraction methods to optimize detection of ZIKV in various body fluids using RT-qPCR as a means of improving the analytical sensitivity of detection. We optimized methods for ZIKV RNA recovery from a number of body fluids by spiking with three varying concentrations of virus, then comparing recovery with that of spiked buffer control. RNA extraction protocols were adjusted as necessary for maximum RNA recovery. Adjustment of the elution step was essential for improved ZIKV RNA recovery from whole blood, saliva, vaginal secretions, and breast milk. Optimal recovery from urine samples required the addition of Urine Conditioning Buffer, and the use of RLT Plus buffer and RNeasy Mini Spin Columns was necessary for RNA extractions from semen samples. Optimized QIAamp MinElute Virus Spin Kit (QIAGEN, Valencia, CA) protocol followed by the singleplex ZIKV RT-qPCR assay provided a reliable method for detection of ZIKV RNA in a variety of biological samples. Improved diagnostics are crucial for timely detection and diagnosis, particularly during pregnancy when the consequences of ZIKV infection can greatly impact the developing fetus.
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spelling pubmed-63676322019-02-13 Optimizing PCR Detection of Zika Virus from Various Body Fluids Gorchakov, Rodion Berry, Rebecca M. Patel, Shital M. El Sahly, Hana M. Ronca, Shannon E. Murray, Kristy O. Am J Trop Med Hyg Articles Current diagnostic protocols of acute Zika virus (ZIKV) infection focus on detection of viral RNA in serum or urine using reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, detecting infection can be a challenge, given that 80% of people with acute ZIKV infection are asymptomatic, and the window to detect viremia in serum is short. The ability to extend that window is needed to detect ZIKV at later time points after infection, particularly in high-risk individuals such as pregnant women. We evaluated RNA extraction methods to optimize detection of ZIKV in various body fluids using RT-qPCR as a means of improving the analytical sensitivity of detection. We optimized methods for ZIKV RNA recovery from a number of body fluids by spiking with three varying concentrations of virus, then comparing recovery with that of spiked buffer control. RNA extraction protocols were adjusted as necessary for maximum RNA recovery. Adjustment of the elution step was essential for improved ZIKV RNA recovery from whole blood, saliva, vaginal secretions, and breast milk. Optimal recovery from urine samples required the addition of Urine Conditioning Buffer, and the use of RLT Plus buffer and RNeasy Mini Spin Columns was necessary for RNA extractions from semen samples. Optimized QIAamp MinElute Virus Spin Kit (QIAGEN, Valencia, CA) protocol followed by the singleplex ZIKV RT-qPCR assay provided a reliable method for detection of ZIKV RNA in a variety of biological samples. Improved diagnostics are crucial for timely detection and diagnosis, particularly during pregnancy when the consequences of ZIKV infection can greatly impact the developing fetus. The American Society of Tropical Medicine and Hygiene 2019-02 2018-12-17 /pmc/articles/PMC6367632/ /pubmed/30560770 http://dx.doi.org/10.4269/ajtmh.18-0755 Text en © The American Society of Tropical Medicine and Hygiene This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Articles
Gorchakov, Rodion
Berry, Rebecca M.
Patel, Shital M.
El Sahly, Hana M.
Ronca, Shannon E.
Murray, Kristy O.
Optimizing PCR Detection of Zika Virus from Various Body Fluids
title Optimizing PCR Detection of Zika Virus from Various Body Fluids
title_full Optimizing PCR Detection of Zika Virus from Various Body Fluids
title_fullStr Optimizing PCR Detection of Zika Virus from Various Body Fluids
title_full_unstemmed Optimizing PCR Detection of Zika Virus from Various Body Fluids
title_short Optimizing PCR Detection of Zika Virus from Various Body Fluids
title_sort optimizing pcr detection of zika virus from various body fluids
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367632/
https://www.ncbi.nlm.nih.gov/pubmed/30560770
http://dx.doi.org/10.4269/ajtmh.18-0755
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