Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen.
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.
College of Life Sciences, Zhejiang Sci-Tech University, 2nd street, Xiasha, Hangzhou, 310018, China.
Juhui
J
Cheng
Yonghou
Y
Jiang
Pinbin
P
Rao
Haigang
H
Wu
Qinfang
Q
Dong
Zhiyi
Z
Wu
Xianfeng
X
Ding
Jiangfeng
J
Guo
D017931:DNA Primers / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010935:Plant Diseases / Q000821:virology; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000821:virology; D013194:Staining and Labeling / Q000379:methods; D044366:Transition Temperature; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D005456:Fluorescent Dyes