A duplex, SYBR Green I-based RT-qPCR assay for the simultaneous detection of Apple chlorotic leaf spot virus and Cherry green ring mottle virus in peach

Abstract

BackgroundCo-infections of Apple chlorotic leaf spot virus (ACLSV) and Cherry green ring mottle virus (CGRMV) in peach is common in China and have resulted in significant yield reductions. A reliable, sensitive and quantitive method is needed to detect and distinguish between ACLSV and CGRMV in peach.FindingsWe developed a sensitive and specific SYBR Green-I based RT-qPCR for the quantification of ACLSV and CGRMV in different peach tissues, and a duplex RT-qPCR system to detect ACLSV and CGRMV simultaneously. The RT-qPCR method was optimized using standard samples transcribed by the T7 Large Scale RNA Production System in vitro. The peach genes, RNA Polymerase subunit II (RPII) and Ubiquitin 10 (UBQ10), which were used as the internal controls for the quantification assay also showed good expression stability in this system. Single RT-qPCR assays showed that CGRMV in peach accumulates to a higher level than ACLSV. The detection limits of the duplex RT-qPCR assay were 102 and 104 copies for ACLSV and CGRMV, respectively. The sensitivity of the duplex RT-qPCR was as high as RT-qPCR and higher than RT-PCR.ConclusionsThe SYBR Green-I RT-qPCR assay provided a sensitive, specific and reliable method for the detection and quantification of ACLSV and CGRMV in different peach tissues. The duplex RT-qPCR system provided a sensitive and specific method to detect and differentiate between ACLSV and CGRMV in a single sample. This RT-qPCR assay could be a useful tool for the routine diagnosis of these two viruses and for disease epidemiology studies in peach orchards.

Natural variation of OsML1, a mitochondrial transcription termination factor, contributes to mesocotyl length variation in rice

“…Total RNA was extracted by the Trizol method (Li et al., 2009). A qRT‐PCR was performed as previously described (Zhao et al., 2013), The primers used are listed in Table S9.…”

Development of a duplex SYBR Green I-based quantitative real-time PCR assay for the rapid differentiation of goose and Muscovy duck parvoviruses

“…The method relying on a SYBR Green fluorescent quantitative PCR was also applied for detection and quantification of other viruses [23, 29]. Furthermore, it has been used to distinguish different virus DNAs in one reaction based on the peaks in the melting curves resulting from the difference in the T m values of the PCR products [1, 32]. Here, we developed a duplex SYBR Green-I based quantitative real-time PCR to rapidly and specifically amplify a region of the GPV and MDPV genome that can be used to distinguish infection by either virus in a single reaction.…”

Survey of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus incidence in Korea by Duplex RT-PCR

“…Multiplex RT-PCR methods are rapid, reliable, and sensitive, and have been widely used to identify more than one virus simultaneously in a single assay, for instance in viral infections of stone fruit ( Sanchez-Navarro et al, 2005 ; Jarosová and Kundu 2010 ; Yardimci and Çulal-Klllç, 2011 ) and lily ( Kwon et al, 2013 ). Multiplex RT-PCR has also been applied to the detection of CNRMV and/or CGRMV ( Noorani et al, 2013 ; Zhao et al, 2013 ), including in sweet cherries. For CNRMV, specific primers were designed based on the replicase gene and have identified stone fruit viruses such as Cherry virus A , Little cherry virus -1, and Prune necrotic ring spot virus , in addition to CNRMV ( Noorani et al, 2013 ).…”

“…For CNRMV, specific primers were designed based on the replicase gene and have identified stone fruit viruses such as Cherry virus A , Little cherry virus -1, and Prune necrotic ring spot virus , in addition to CNRMV ( Noorani et al, 2013 ). A duplex RT-qPCR method was also developed for Apple chlorotic leaf spot virus and CGRMV in peach ( Zhao et al, 2013 ).…”