NDLI: Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen

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Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen

Content Provider	Springer Nature Link
Author	Cheng, Juhui Jiang, Yonghou Rao, Pinbin Wu, Haigang Dong, Qinfang Wu, Zhiyi Ding, Xianfeng Guo, Jiangfeng
Copyright Year	2012
Abstract	SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye. Preferential binding of SG during PCR and inhibition of PCR often result in failure to detect multiple amplicons in multiplex reactions. In the present study, a novel single-tube, multiplex real-time PCR with EvaGreen dye (EG) was developed and evaluated for simultaneous detection of pathogenic targets by using five potato viruses as models. The PCR products obtained using five sets of specific primers were analyzed by melting curve analysis. The assay could specifically detect and differentiate the five potato viruses by producing a distinct peak for each amplification product and exhibited a high reproducibility with coefficients of variation from 0.01 to 0.25 %. Detection sensitivity of the assay ranged from 100 to 500 copies/μL for each virus. The results of this study demonstrate that multiplex real-time PCR and melting-curve analysis with EG is a sensitive, specific and inexpensive method for simultaneous detection of multiple pathogens.
Starting Page	379
Ending Page	386
Page Count	8
File Format	PDF
ISSN	03048608
Journal	Archives of Virology
Volume Number	158
Issue Number	2
e-ISSN	14328798
Language	English
Publisher	Springer Vienna
Publisher Date	2012-10-10
Publisher Place	Vienna
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Virology Medical Microbiology Infectious Diseases
Content Type	Text
Resource Type	Article
Subject	Virology Medicine

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