Cargando…

Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

BACKGROUND: Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and sym...

Descripción completa

Detalles Bibliográficos
Autores principales: Pang, Zheng, Li, Aqian, Li, Jiandong, Qu, Jing, He, Chengcheng, Zhang, Shuo, Li, Chuan, Zhang, Quanfu, Liang, Mifang, Li, Dexin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3994070/
https://www.ncbi.nlm.nih.gov/pubmed/24752452
http://dx.doi.org/10.1371/journal.pone.0095635
_version_ 1782312662248456192
author Pang, Zheng
Li, Aqian
Li, Jiandong
Qu, Jing
He, Chengcheng
Zhang, Shuo
Li, Chuan
Zhang, Quanfu
Liang, Mifang
Li, Dexin
author_facet Pang, Zheng
Li, Aqian
Li, Jiandong
Qu, Jing
He, Chengcheng
Zhang, Shuo
Li, Chuan
Zhang, Quanfu
Liang, Mifang
Li, Dexin
author_sort Pang, Zheng
collection PubMed
description BACKGROUND: Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. RESULTS: Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. CONCLUSIONS: Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs.
format Online
Article
Text
id pubmed-3994070
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-39940702014-04-25 Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses Pang, Zheng Li, Aqian Li, Jiandong Qu, Jing He, Chengcheng Zhang, Shuo Li, Chuan Zhang, Quanfu Liang, Mifang Li, Dexin PLoS One Research Article BACKGROUND: Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. RESULTS: Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. CONCLUSIONS: Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs. Public Library of Science 2014-04-21 /pmc/articles/PMC3994070/ /pubmed/24752452 http://dx.doi.org/10.1371/journal.pone.0095635 Text en © 2014 Pang et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Pang, Zheng
Li, Aqian
Li, Jiandong
Qu, Jing
He, Chengcheng
Zhang, Shuo
Li, Chuan
Zhang, Quanfu
Liang, Mifang
Li, Dexin
Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses
title Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses
title_full Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses
title_fullStr Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses
title_full_unstemmed Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses
title_short Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses
title_sort comprehensive multiplex one-step real-time taqman qrt-pcr assays for detection and quantification of hemorrhagic fever viruses
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3994070/
https://www.ncbi.nlm.nih.gov/pubmed/24752452
http://dx.doi.org/10.1371/journal.pone.0095635
work_keys_str_mv AT pangzheng comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT liaqian comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT lijiandong comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT qujing comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT hechengcheng comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT zhangshuo comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT lichuan comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT zhangquanfu comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT liangmifang comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses
AT lidexin comprehensivemultiplexonesteprealtimetaqmanqrtpcrassaysfordetectionandquantificationofhemorrhagicfeverviruses