Development and Application of RT-qPCR Assay for Detection of Potato virus S by Universal Detection System

doi:10.7668/hbnxb.20191131

Abstract

Abstract: In order to identify PVS and its individual strains accurately and quickly, and understand the content of PVS in potato leaves, petioles, stems, roots and dormant tubers, which is helpful for selecting suitable detection sites, an Reverse transcription-qPCR (RT-qPCR) was developed by designing a pair of universal primer, and PVS^O and PVS^A strains were identified using melting curve. The specificity of the system was evaluated with Potato virus X (PVX), Potato virus Y (PVY), Potato virus M (PVM), Potato virus A (PVA) and Potato leafroll virus (PLRV) positive samples. The standard curve of PVS^O and PVS^A were established with PVS^O and PVS^A standard recombinant plasmid, and the contents of PVS^O and PVS^A in potato leaves, petioles, stems, roots and dormant tubers of potato varieties Youjin and Jizhangshu 12 inoculated with PVS^O and PVS^A were detected. Moreover, ninety positive samples collected from the 11 Provinces (municipality or autonomous region) were verified by the RT-qPCR system. The amplification of PVS^O and PVS^A strains showed specific peak at 85.77-86.00℃ and 87.78-87.91℃, respectively, in melting curve analysis, but no cross-reaction was found for PVX, PVY, PVM, PVA and PLRV samples. When the concentration of PVS^O and PVS^A recombinant plasmid were from 1.09×10⁵-1.09×10⁹ copies/μL and 1.26×10⁵-1.26×10⁹ copies/μL, respectively, there was a good linear relationship between the standard curve circulation threshold (Ct) and the log value of PVS virus particles, with determination coefficient being 0.994 2 and 0.991 2, respectively. The content of PVS^O and PVS^A in five parts of potato tissues were over 10⁷, and all of them could be detected, with the content of PVS^O being the highest in petioles. Ninety PVS positive samples from 11 Provinces (municipality or autonomous region) were detected effectively. The PVS RT-qPCR detection system established was fast, accurate and specific and PVS^O and PVS^A strains could be identified by specific peak in melting curve. The five parts of tissue including leaves, petioles, stems, roots and dormant tubers could be used for detection, with the advantage of good practicability. This research provides technical support to produce virus-free seed potatoes.

Key words: Potato virus S, Strains, RT-qPCR, Detection, Seed potato

CLC Number:

S532
S432.4

CHENG Shengqun, Lü Wenhe, GAO Yanling, BAI Yanju, FAN Guoquan, ZHANG Wei, ZHANG Shu, QIU Cailing, SHEN Yu, DONG Xuezhi, BAI Yamei. Development and Application of RT-qPCR Assay for Detection of Potato virus S by Universal Detection System[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(6): 187-194. doi: 10.7668/hbnxb.20191131.

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