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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
Zhang, Ni Liu, Zhengwen Han, Qunying Qiu, Jianming Chen, Jinghong Zhang, Guoyu Li, Zhu Lou, Sai Li, Na
Zhang, Ni
Liu, Zhengwen
Han, Qunying
Qiu, Jianming
Chen, Jinghong
Zhang, Guoyu
Li, Zhu
Lou, Sai
Li, Na
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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.
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Date
2011-07-29
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BioMed Central
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Zhang_et_al_2011.pdf
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Keywords
Bovine viral diarrhea virus type-1, cRNA standard, SYBR Green I RT-PCR, Quantitation, Cell culture
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.