A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.
Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of po...
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ftdoajarticles:oai:doaj.org/article:29f3c6957434413bbed1c1164d3cdd71 2023-05-15T15:17:40+02:00 A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses. Ashutosh Wadhwa Kimberly Wilkins Jinxin Gao Rene Edgar Condori Condori Crystal M Gigante Hui Zhao Xiaoyue Ma James A Ellison Lauren Greenberg Andres Velasco-Villa Lillian Orciari Yu Li 2017-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0005258 https://doaj.org/article/29f3c6957434413bbed1c1164d3cdd71 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC5230753?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0005258 https://doaj.org/article/29f3c6957434413bbed1c1164d3cdd71 PLoS Neglected Tropical Diseases, Vol 11, Iss 1, p e0005258 (2017) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2017 ftdoajarticles https://doi.org/10.1371/journal.pntd.0005258 2022-12-31T12:48:21Z Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 11 1 e0005258 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Ashutosh Wadhwa Kimberly Wilkins Jinxin Gao Rene Edgar Condori Condori Crystal M Gigante Hui Zhao Xiaoyue Ma James A Ellison Lauren Greenberg Andres Velasco-Villa Lillian Orciari Yu Li A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which ... |
format |
Article in Journal/Newspaper |
author |
Ashutosh Wadhwa Kimberly Wilkins Jinxin Gao Rene Edgar Condori Condori Crystal M Gigante Hui Zhao Xiaoyue Ma James A Ellison Lauren Greenberg Andres Velasco-Villa Lillian Orciari Yu Li |
author_facet |
Ashutosh Wadhwa Kimberly Wilkins Jinxin Gao Rene Edgar Condori Condori Crystal M Gigante Hui Zhao Xiaoyue Ma James A Ellison Lauren Greenberg Andres Velasco-Villa Lillian Orciari Yu Li |
author_sort |
Ashutosh Wadhwa |
title |
A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses. |
title_short |
A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses. |
title_full |
A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses. |
title_fullStr |
A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses. |
title_full_unstemmed |
A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses. |
title_sort |
pan-lyssavirus taqman real-time rt-pcr assay for the detection of highly variable rabies virus and other lyssaviruses. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2017 |
url |
https://doi.org/10.1371/journal.pntd.0005258 https://doaj.org/article/29f3c6957434413bbed1c1164d3cdd71 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 11, Iss 1, p e0005258 (2017) |
op_relation |
http://europepmc.org/articles/PMC5230753?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0005258 https://doaj.org/article/29f3c6957434413bbed1c1164d3cdd71 |
op_doi |
https://doi.org/10.1371/journal.pntd.0005258 |
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PLOS Neglected Tropical Diseases |
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11 |
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1 |
container_start_page |
e0005258 |
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1766347914052894720 |