Establishment of a Triple RT-qPCR Method for the Simultaneous Detecting of PMWaV-1, PMWaV-2 and PMWaV-3

doi:10.7668/hbnxb.2017.02.006

Abstract

Abstract: The objective of this paper was to provide a technique for quantitative and quanlitative studies of PMWaV by establishing the method of Real-time Fluorescent Quantitative RT-PCR (RT-qPCR)for simultaneous quantitative detection of PMWaV-1,PMWaV-2 and PMWaV-3.Primers and probes were designed and synthesized according to the CP gene of PMWaV-1,PMWaV-2 and PMWaV-3,respectively.After optimized the reaction system of triple RT-qPCR,the standard curves were generated by purified DNA of the recombined plasmid of PMWaV-1,PMWaV-2,PMWaV-3 coat protein gene,respectively.And the method of RT-qPCR for the simultaneous detection of PMWaV-1,PMWaV-2,PMWaV-3 had been improved.The amplification efficiency of PMWaV-1,PMWaV-2,PMWaV-3 in the RT-qPCR method was up to 101.7%,97.1%,106.7%,respectively.Meanwhile,with the R² between 0.996 and 0.999.The sensibility test of triple RT-qPCR syetem showed the detection limit of quantification for PMWaV-1,PMWaV-2,PMWaV-3 was 99,98 and 868 copies,respectively.Moreover,repeats experiment datas showed that standard deviation was below 0.26 and variable coefficient was below 1.44%.This study indicated that the method for simultaneous detection and quantification of PMWaV-1,PMWaV-2,PMWaV-3 was a fast,sably and sensitive reaction system,which could be the technological base for quantitative and quanlitative studies of PMWaV.

Key words: Mealybug Wilt of Pineapple, Dctection method, RT-qPCR, PMWaV, TaqMan probe

CLC Number:

S432.4
Q78

HU Jiayi, LUO Zhiwen, HE Fan, LI Xianghong, HU Fuchu, FAN Hongyan, LIU Zhixin, ZHANG Zhili. Establishment of a Triple RT-qPCR Method for the Simultaneous Detecting of PMWaV-1, PMWaV-2 and PMWaV-3[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(2): 38-44. doi: 10.7668/hbnxb.2017.02.006.

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