title: Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR.
abstract: BACKGROUND
  Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle and acts as a surrogate model for hepatitis C virus (HCV). One-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-PCR) assay based on SYBR Green I dye has not been established for BVDV detection. This study aims to develop a quantitative one-step RT-PCR assay to detect BVDV type-1 in cell culture.
  
  
  RESULTS
  One-step quantitative SYBR Green I RT-PCR was developed by amplifying cDNA template from viral RNA and using in vitro transcribed BVDV RNA to establish a standard curve. The assay had a detection limit as low as 100 copies/ml of BVDV RNA, a reaction efficiency of 103.2%, a correlation coefficient (R2) of 0.995, and a maximum intra-assay CV of 2.63%. It was 10-fold more sensitive than conventional RT-PCR and can quantitatively detect BVDV RNA levels from 10-fold serial dilutions of titrated viruses containing a titer from 10(-1) to 10(-5) TCID50, without non-specific amplification. Melting curve analysis showed no primer-dimers and non-specific products.
  
  
  CONCLUSIONS
  The one-step SYBR Green I RT-PCR is specific, sensitive and reproducible for the quantification of BVDV in cell culture. This one-step SYBR Green I RT-PCR strategy may be further optimized as a reliable assay for diagnosing and monitoring BVDV infection in animals. It may also be applied to evaluate candidate agents against HCV using BVDV cell culture model.
authors.affiliation: Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, the People's Republic of China.
authors.forename: Ni
authors.initials: N
authors.lastname: Zhang
authors.forename: Zhengwen
authors.initials: Z
authors.lastname: Liu
authors.forename: Qunying
authors.initials: Q
authors.lastname: Han
authors.forename: Jianming
authors.initials: J
authors.lastname: Qiu
authors.forename: Jinghong
authors.initials: J
authors.lastname: Chen
authors.forename: Guoyu
authors.initials: G
authors.lastname: Zhang
authors.forename: Zhu
authors.initials: Z
authors.lastname: Li
authors.forename: Sai
authors.initials: S
authors.lastname: Lou
authors.forename: Na
authors.initials: N
authors.lastname: Li
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D018929:Cell Culture Techniques; D003959:Diamines; D030241:Diarrhea Virus 1, Bovine Viral / Q000302:isolation & purification*; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D019562:Viral Load / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I