dc.contributor.author | Zhang, Ni | |
dc.contributor.author | Liu, Zhengwen | |
dc.contributor.author | Han, Qunying | |
dc.contributor.author | Qiu, Jianming | |
dc.contributor.author | Chen, Jinghong | |
dc.contributor.author | Zhang, Guoyu | |
dc.contributor.author | Li, Zhu | |
dc.contributor.author | Lou, Sai | |
dc.contributor.author | Li, Na | |
dc.date.accessioned | 2014-04-11T17:47:36Z | |
dc.date.available | 2014-04-11T17:47:36Z | |
dc.date.issued | 2011-07-29 | |
dc.identifier.citation | Ni Zhang, Zhengwen Liu, Qunying Han, Jianming Qiu, Jinghong Chen, Guoyu Zhang, Zhu Li, Sai Lou, Na Li. "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." 2011. Virology Journal 8:374. http://www.dx.doi.org/10.1186/1743-422X-8-374. | |
dc.identifier.uri | http://hdl.handle.net/1808/13456 | |
dc.description.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. | |
dc.publisher | BioMed Central | |
dc.subject | Bovine viral diarrhea virus type-1 | |
dc.subject | cRNA standard | |
dc.subject | SYBR Green I RT-PCR | |
dc.subject | Quantitation | |
dc.subject | Cell culture | |
dc.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 | |
dc.type | Article | |
kusw.kuauthor | Qiu, Jianming | |
kusw.kudepartment | Molecular Biosciences | |
kusw.oastatus | fullparticipation | |
dc.identifier.doi | 10.1186/1743-422X-8-374 | |
kusw.oaversion | Scholarly/refereed, publisher version | |
kusw.oapolicy | This item meets KU Open Access policy criteria. | |
dc.rights.accessrights | openAccess | |