title: Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.
abstract: 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.
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Pang
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Aqian
authors.initials: A
authors.lastname: Li
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Jiandong
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Qu
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Chengcheng
authors.initials: C
authors.lastname: He
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Li
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Quanfu
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Mifang
authors.initials: M
authors.lastname: Liang
authors.affiliation: Key Laboratory of Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Dexin
authors.initials: D
authors.lastname: Li
mesh_terms: D017931:DNA Primers / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014780:Viruses / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
references.citation: Med Trop (Mars). 2003;63(3):296-9
references.pmid: 14579470
references.citation: Curr Opin Immunol. 2005 Aug;17(4):399-403
references.pmid: 15955687
references.citation: Sci Am. 1995 Oct;273(4):56-64
references.pmid: 7481722
references.citation: MMWR Morb Mortal Wkly Rep. 2001 Feb 9;50(5):73-7
references.pmid: 11686289
references.citation: Euro Surveill. 2004 Dec 15;9(12):E11-2
references.pmid: 15677844
references.citation: J Clin Virol. 2004 May;30(1):94-9
references.pmid: 15072761
references.citation: Am J Trop Med Hyg. 1997 Nov;57(5):519-25
references.pmid: 9392589
references.citation: Nucleic Acids Res. 2002 Mar 15;30(6):1292-305
references.pmid: 11884626
references.citation: Clin Microbiol Rev. 2006 Jan;19(1):165-256
references.pmid: 16418529
references.citation: Antiviral Res. 2004 Dec;64(3):145-60
references.pmid: 15550268
references.citation: PLoS Pathog. 2009 May;5(5):e1000455
references.pmid: 19478873
references.citation: Nat Med. 2004 Dec;10(12 Suppl):S110-21
references.pmid: 15577929
references.citation: Vector Borne Zoonotic Dis. 2007 Winter;7(4):467-77
references.pmid: 18020965
references.citation: Am J Trop Med Hyg. 2010 May;82(5):954-60
references.pmid: 20439981
references.citation: N Engl J Med. 2012 Aug 30;367(9):834-41
references.pmid: 22931317
references.citation: PLoS Pathog. 2012 Sep;8(9):e1002924
references.pmid: 23028323
references.citation: Antiviral Res. 2003 Jan;57(1-2):61-87
references.pmid: 12615304
references.citation: PLoS Pathog. 2008 Apr 18;4(4):e1000047
references.pmid: 18421377
references.citation: J Virol Methods. 2005 Mar;124(1-2):21-6
references.pmid: 15664046
references.citation: J Pharm Pract. 2012 Oct;25(5):521-9
references.pmid: 23011963
references.citation: J Clin Microbiol. 2002 Jul;40(7):2323-30
references.pmid: 12089242