title: Development of a SYBR-Green Ⅰ quantitative PCR assay for the detection and genotyping of different hantaviruses.
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.
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Ziyu
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of General Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Yuan
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Qikang
authors.initials: Q
authors.lastname: Ying
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Lan
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Linfeng
authors.initials: L
authors.lastname: Cheng
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Fanglin
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of General Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Lu
authors.affiliation: Department of Microbiology, The Fourth Military Medical University, Xi'an, Shaanxi, P.R. China.
authors.forename: Xing'an
authors.initials: X
authors.lastname: Wu
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D003959:Diamines; D018628:Gene Dosage; D016679:Genome, Viral / Q000235:genetics; D005838:Genotype; D018063:Hantaan virus / Q000235:genetics* / Q000502:physiology; D006480:Hemorrhagic Fever with Renal Syndrome / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D051379:Mice; D009930:Organic Chemicals / Q000737:chemistry*; D011804:Quinolines; D018075:RNA, Complementary / Q000097:blood / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000097:blood / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D029263:Seoul virus / Q000235:genetics* / Q000502:physiology; D013045:Species Specificity
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D018075:RNA, Complementary; D012367:RNA, Viral; C098022:SYBR Green I