Optimization and Application of Real-time Fluorescence Quantitative PCR for Detection of Ectropis obliqua Nucleopolyhedrovirus

YUAN Zhi-jun; ZHANG Chuan-xi; XIAO Qiang; YIN Kun-shan

doi:10.13305/j.cnki.jts.2013.03.006

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Journal of Tea Science ›› 2013, Vol. 33 ›› Issue (3) : 229-236. DOI: 10.13305/j.cnki.jts.2013.03.006

Optimization and Application of Real-time Fluorescence Quantitative PCR for Detection of Ectropis obliqua Nucleopolyhedrovirus

YUAN Zhi-jun¹, ZHANG Chuan-xi², XIAO Qiang^1,*, YIN Kun-shan¹

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Abstract

Primers were designed based on p16 gene from Ectropis obliqua nucleopolyhedrovirus in order to optimize the detection method of SYBR Green I real-time fluorescence quantitation PCR for EoNPV, and the standard curve of SYBR Green I real-time fluorescence quantitative PCR for p16 gene was established using the recombinant plasmid DNA as template. Statistic analysis showed that there was a good linear relationship between Ct value and the logarithmic value of plasmid concentrations (R²=0.9981). The sensitivity of the method was 10² copies/μl, and wide detection range of 6 orders of magnitude was obtained. Larave infected at different time were sampled and detected by the method, and the results showed that there was a good linear relation between the logarithmic value of the multiples of gene copies and the infection time (R²=0.9935). Experimental results indicated that the method can identify biological pesticides from qualitative and quantitative aspects, and distinguish the high homology nuclearpolyhedrosisvirus (Euproctis pseudoconspersa nuclearpolyhedrosisvirus) from EoNPV accurately.

Key words

detection / qualitative and quantitative / SYBR Green I / EoNPV

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YUAN Zhi-jun, ZHANG Chuan-xi, XIAO Qiang, YIN Kun-shan. Optimization and Application of Real-time Fluorescence Quantitative PCR for Detection of Ectropis obliqua Nucleopolyhedrovirus[J]. Journal of Tea Science. 2013, 33(3): 229-236 https://doi.org/10.13305/j.cnki.jts.2013.03.006

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