One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees

Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay

“…), for viroids and phytoplasmas of pome fruit trees (Malandraki et al . ), for the Grapevine virus A , B , and D (Osman et al . ), for the PMTV and for the TRV (Mumford et al .…”

“…This manuscript describes the establishment and application of a reliable and simple multiplex TaqMan RT-qPCR assay for the simultaneous detection of the NYSV and the NMV in narcissus samples. Prior to this study, probe-based detection methods have been described in other studies, including the NYSV 10 À3 10 À8 10 3 10 À8 10 3 NMV 10 À3 10 À8 10 3 10 À8 10 3 multiplex simultaneous detection assay for Citrus viruses (Osman et al 2015), for diseases and pathogens of potato tubers (Qu et al 2011), for viroids and phytoplasmas of pome fruit trees (Malandraki et al 2015), for the Grapevine virus A, B, and D (Osman et al 2013), for the PMTV and for the TRV (Mumford et al 2000). These studies have demonstrated the detection superiority of qPCR over other detection technologies, such as the salient feature of high sensitivity (Fu et al 2015).…”

“…The plasmid copy numbers were calculated using equation copies/ng = (N A × A)/( n × mw) as described by Malandraki et al . ().…”

“…The plasmid containing the partial CP gene concentration was quantified via Thermo NanoDrop 2000c (Thermo Scientific, Wilmington, DE). The plasmid copy numbers were calculated using equation copies/ng = (N A 9 A)/(n 9 mw) as described by Malandraki et al (2015).…”

“…Consequently, assays based on RT-PCR methods were established to detect Narcissus latent virus (NLV), NYSV and NMV in culture fields (Berniak et al 2013;Hori et al 2013). Multiplex real-time RT-PCR in particular has been applied to facilitate the detection of target viruses due to high precision and sensitivity, as well as due to fast and simple performance for the detection of several pathogens in a single reaction (Heid et al 1996;Qu et al 2011;Malandraki et al 2015;Wang et al 2016). Accordingly, the common multiplex real-time RT-PCR method was developed for plant material that was infected with multiple viruses, for example, the potato with the Potato mop top virus (PMTV) and the Tobacco rattle virus (TRV) (Mumford et al 2000), and citrus with the Citrus tristeza virus, the Citrus psorosis virus and the Citrus leaf blotch virus (Osman et al 2015).…”