Development of a SYBR-Green Ⅰ quantitative PCR assay for the detection and genotyping of different hantaviruses

Liu,Ziyu; Wang,Fang; Yuan,Lijuan; Zhang,Xiaoxiao; Ying,Qikang; Yu,Lan; Zhang,Liang; Cheng,Linfeng; Zhang,Fanglin; Lu,Jianguo; Wu,Xing'an

doi:10.3892/ijmm.2016.2678

Development of a SYBR-Green Ⅰ quantitative PCR assay for the detection and genotyping of different hantaviruses

Authors:
- Ziyu Liu
- Fang Wang
- Lijuan Yuan
- Xiaoxiao Zhang
- Qikang Ying
- Lan Yu
- Liang Zhang
- Linfeng Cheng
- Fanglin Zhang
- Jianguo Lu
- Xing'an Wu
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Affiliations: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China, Department of General Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China
Published online on: July 13, 2016 https://doi.org/10.3892/ijmm.2016.2678
Pages: 951-960

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Abstract

Hemorrhagic fever with renal syndrome (HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus (HTNV) and the Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBR‑Green I-based reverse transcription-quantitiative polymerase chain reaction (RT-qPCR) assay, which targeted the S gene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold (Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient (R2) of 0.994, efficiency of amplification (E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2 of 0.993, E of 104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101 copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBR‑Green I-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases.

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1	Schmaljohn CS, Hasty SE, Harrison SA and Dalrymple JM: Characterization of Hantaan virions, the prototype virus of hemorrhagic fever with renal syndrome. J Infect Dis. 148:1005–1012. 1983. View Article : Google Scholar : PubMed/NCBI
2	Jonsson CB, Figueiredo LT and Vapalahti O: A global perspective on hantavirus ecology, epidemiology, and disease. Clin Microbiol Rev. 23:412–441. 2010. View Article : Google Scholar : PubMed/NCBI
3	Vaheri A, Henttonen H, Voutilainen L, Mustonen J, Sironen T and Vapalahti O: Hantavirus infections in Europe and their impact on public health. Rev Med Virol. 23:35–49. 2013. View Article : Google Scholar
4	Roda Gracia J, Schumann B and Seidler A: Climate variability and the occurrence of human puumala hantavirus infections in Europe: a systematic review. Zoonoses Public Health. 62:465–478. 2015. View Article : Google Scholar : PubMed/NCBI
5	Maes P, Clement J, Gavrilovskaya I and Van Ranst M: Hantaviruses: Immunology, treatment, and prevention. Viral Immunol. 17:481–497. 2004. View Article : Google Scholar
6	Kanerva M, Mustonen J and Vaheri A: Pathogenesis of puumala and other hantavirus infections. Rev Med Virol. 8:67–86. 1998. View Article : Google Scholar
7	Khaiboullina SF, Morzunov SP and St Jeor SC : Hantaviruses: molecular biology, evolution and pathogenesis. Curr Mol Med. 5:773–790. 2005. View Article : Google Scholar : PubMed/NCBI
8	Clement J, Heyman P, McKenna P, Colson P and Avsic-Zupanc T: The hantaviruses of Europe: from the bedside to the bench. Emerg Infect Dis. 3:205–211. 1997. View Article : Google Scholar : PubMed/NCBI
9	Hedman K, Vaheri A and Brummer-Korvenkontio M: Rapid diagnosis of hantavirus disease with an IgG-avidity assay. Lancet. 338:1353–1356. 1991. View Article : Google Scholar : PubMed/NCBI
10	Mohamed N, Nilsson E, Johansson P, Klingström J, Evander M, Ahlm C and Bucht G: Development and evaluation of a broad reacting SYBR-green based quantitative real-time PCR for the detection of different hantaviruses. J Clin Virol. 56:280–285. 2013. View Article : Google Scholar : PubMed/NCBI
11	Garin D, Peyrefitte C, Crance JM, Le Faou A, Jouan A and Bouloy M: Highly sensitive Taqman PCR detection of Puumala hantavirus. Microbes Infect. 3:739–745. 2001. View Article : Google Scholar : PubMed/NCBI
12	Dash PK, Boutonnier A, Prina E, Sharma S and Reiter P: Development of a SYBR-Green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti. Virol J. 9:272012. View Article : Google Scholar
13	Jiang W, Yu HT, Zhao K, Zhang Y, Du H, Wang PZ and Bai XF: Quantification of Hantaan virus with a SYBR-Green I-based one-step qRT-PCR assay. PLoS One. 8:e815252013. View Article : Google Scholar
14	Yi J, Xu Z, Zhuang R, Wang J, Zhang Y, Ma Y, Liu B, Zhang Y, Zhang C, Yan G, et al: Hantaan virus RNA load in patients having hemorrhagic fever with renal syndrome: correlation with disease severity. J Infect Dis. 207:1457–1461. 2013. View Article : Google Scholar
15	Wei F, Li JL, Ling JX, Chen LJ, Li N, Liu YY, Luo F, Xiong HR, Hou W and Yang ZQ: Establishment of SYBR-Green-based qPCR assay for rapid evaluation and quantification for anti-Hantaan virus compounds in vitro and in suckling mice. Virus Genes. 46:54–62. 2013. View Article : Google Scholar
16	Schmaljohn C and Hjelle B: Hantaviruses: a global disease problem. Emerg Infect Dis. 3:95–104. 1997. View Article : Google Scholar : PubMed/NCBI
17	Bird BH, Bawiec DA, Ksiazek TG, Shoemaker TR and Nichol ST: Highly sensitive and broadly reactive quantitative reverse transcription-PCR assay for high-throughput detection of Rift Valley fever virus. J Clin Microbiol. 45:3506–3513. 2007. View Article : Google Scholar : PubMed/NCBI
18	Sall AA, Macondo EA, Sène OK, Diagne M, Sylla R, Mondo M, Girault L, Marrama L, Spiegel A, Diallo M, et al: Use of reverse transcriptase PCR in early diagnosis of Rift Valley fever. Clin Diagn Lab Immunol. 9:713–715. 2002.PubMed/NCBI
19	Drosten C, Göttig S, Schilling S, Asper M, Panning M, Schmitz H and Günther S: Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol. 40:2323–2330. 2002. View Article : Google Scholar : PubMed/NCBI
20	Jiang W, Wang PZ, Yu HT, Zhang Y, Zhao K, Du H and Bai XF: Development of a SYBR-Green I based one-step real-time PCR assay for the detection of Hantaan virus. J Virol Methods. 196:145–151. 2014. View Article : Google Scholar
21	Chen H, Parimelalagan M, Lai YL, Lee KS, Koay ES, Hapuarachchi HC, Ng LC, Ho PS and Chu JJ: Development and evaluation of a SYBR-Green-based real-time multiplex RT-PCR assay for simultaneous detection and serotyping of dengue and Chikungunya viruses. J Mol Diagn. 17:722–728. 2015. View Article : Google Scholar : PubMed/NCBI