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Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus

BACKGROUND: Research with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infe...

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Autores principales: Stefan, Christopher P., Chase, Kitty, Coyne, Susan, Kulesh, David A., Minogue, Timothy D., Koehler, Jeffrey W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815133/
https://www.ncbi.nlm.nih.gov/pubmed/27029488
http://dx.doi.org/10.1186/s12985-016-0509-3
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author Stefan, Christopher P.
Chase, Kitty
Coyne, Susan
Kulesh, David A.
Minogue, Timothy D.
Koehler, Jeffrey W.
author_facet Stefan, Christopher P.
Chase, Kitty
Coyne, Susan
Kulesh, David A.
Minogue, Timothy D.
Koehler, Jeffrey W.
author_sort Stefan, Christopher P.
collection PubMed
description BACKGROUND: Research with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices. METHODS: Total nucleic acid from virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV. RESULTS: Optimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors. CONCLUSIONS: PTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-016-0509-3) contains supplementary material, which is available to authorized users.
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spelling pubmed-48151332016-04-01 Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus Stefan, Christopher P. Chase, Kitty Coyne, Susan Kulesh, David A. Minogue, Timothy D. Koehler, Jeffrey W. Virol J Research BACKGROUND: Research with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices. METHODS: Total nucleic acid from virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV. RESULTS: Optimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors. CONCLUSIONS: PTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12985-016-0509-3) contains supplementary material, which is available to authorized users. BioMed Central 2016-03-31 /pmc/articles/PMC4815133/ /pubmed/27029488 http://dx.doi.org/10.1186/s12985-016-0509-3 Text en © Stefan et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Stefan, Christopher P.
Chase, Kitty
Coyne, Susan
Kulesh, David A.
Minogue, Timothy D.
Koehler, Jeffrey W.
Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus
title Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus
title_full Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus
title_fullStr Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus
title_full_unstemmed Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus
title_short Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus
title_sort development of real-time reverse transcriptase qpcr assays for the detection of punta toro virus and pichinde virus
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815133/
https://www.ncbi.nlm.nih.gov/pubmed/27029488
http://dx.doi.org/10.1186/s12985-016-0509-3
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