LAMP assay and rapid sample preparation method for on‐site detection of flavescence dorée phytoplasma in grapevine

Abstract

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

“…The performance of the LAMP assay was compared with the currently used qPCR protocols. This protocol includes the newly developed LAMP assay for BNp and the published LAMP assay for FDp (Kogovšek et al 2015). These LAMP assays were shown here to be effective in the detection of both BNp and FDp in grapevine without any DNA extraction step, and with comparable sensitivity to that of qPCR.…”

“…We additionally evaluated the analytical sensitivity of direct testing of leaf-vein homogenates prepared with Ultra Turrax Tube Drive device (UTTD, IKA). This device is favoured due to its cost and performance efficiencies and its on-site applicability for FDp detection (Kogovšek et al 2015). A comparative study was performed on 286 grapevine samples collected during an official survey in the 2015 growing season.…”

“…The LAMP reactions were performed in 8-well strips, or 96-well plate in GenieII (Optigene) or Roche LC480 instruments, respectively. In the 'multi' LAMP (mLAMP) assays where secA (targeting BNp; this study) and 23S rRNA (targeting FDp) (Kogovšek et al 2015) were combined, the same primer and reagent concentrations were used as above. For the LAMP product annealing temperature determination (i.e., Tm), the fluorescence was detected during the cooling of the samples from 98°C to 80°C Genomic DNA from pure cultured fungal isolates was extracted with the NucleoSpin Plant II (Macherey-Nagel) by using the cetyl-trimethylammonium-bromide-based based extraction buffer supplied with this kit (GenieII) or during the heating of the samples from 62°C to 98°C (Roche LC480).…”

“…Symptoms caused by BNp and FDp are not distinguishable by visual inspection and as the distribution of phytoplasma is uneven within a host that has a very low titre (Prezelj et al 2013), only specific molecular approaches are suitable for the accurate and reliable detection of BNp and FDp. In diagnostic laboratories, assays based on real-time PCR (qPCR) are widely used for the detection of BNp and FDp (Angelini et al 2007;Bianco et al 2004;Galetto et al 2005;Hren et al 2007;Margaria et al 2009;Pelletier et al 2009). Furthermore, a protocol for the detection of FDp based on loop-mediated isothermal amplification (LAMP) assay was developed recently (Notomi et al 2000;Kogovšek et al 2015). LAMP is a highly specific and rapid technique, and it also circumvents the sensitivity of PCR and qPCR to inhibitors in plant extracts (Francois et al 2011); furthermore, its isothermal nature provides the potential for it to be deployed in the field (Kogovšek et al 2015;Tomlinson et al 2010a).…”

“…Furthermore, a protocol for the detection of FDp based on loop-mediated isothermal amplification (LAMP) assay was developed recently (Notomi et al 2000;Kogovšek et al 2015). LAMP is a highly specific and rapid technique, and it also circumvents the sensitivity of PCR and qPCR to inhibitors in plant extracts (Francois et al 2011); furthermore, its isothermal nature provides the potential for it to be deployed in the field (Kogovšek et al 2015;Tomlinson et al 2010a). LAMP has shown a comparable or better performance to other detection methods and a wide applicability for the detection of plant pathogenic bacteria (Lenarčič et al 2014), viroids (Lenarčič et al 2013), fungi (Tomlinson et al 2010b) and phytoplasmas (Bekele et al 2011;Dickinson 2015;Hodgetts et al 2011;Kogovšek et al 2015;Tomlinson et al 2010a).…”