Optimization of a Real-Time RT-PCR Assay and its Comparison with ELISA, Conventional RT-PCR and the Grow-out Test for Large Scale Diagnosis of Potato virus Y in Dormant Potato Tubers

Abstract

A TaqMan real-time reverse transcription-PCR (real-time RT-PCR) procedure was developed, optimized, and compared with other routine methods to detect Potato virus Y (PVY) in dormant potato tubers. Three pairs of primers and probes were designed and evaluated for their suitability to facilitate the real-time RT-PCR detection of PVY for all strain groups including PVY O , PVY N , PVY N: O (0 PVY N-Wi ) and PVY NTN . Among the primer and probe combinations tested, the combination PVY-1 produced the lowest threshold cycle (Ct) value of 25.75. The procedure was further optimized by adjusting various parameters including primer/probe concentration, reaction volume, amplification cycles, and master mixes from different sources. The real-time RT-PCR was then employed to detect PVY from dormant tubers of different cultivars and potato fields, and the results were compared with those obtained from conventional RT-PCR, enzyme-linked immunosorbent assay (ELISA) on sprouts and grow-out testing. Out of 1,069 single-virus infected (PVY only) tubers tested, both formats of RT-PCR detected PVY in 52 samples, ELISA on sprouts in 45, ELISA on leaves in 54 and visual observations in 53. However, in 61 multiple-virus infected tubers tested, both formats of RT-PCR, and ELISA on both sprouts and leaves detected a similar number of positives, thus, making all the methods equally sensitive. Considering that ELISA requires sprouting of dormant potato tubers for PVY testing, growout testing takes approximately 6-8 weeks to obtain results, and conventional RT-PCR needs post-PCR processing, realtime RT-PCR offers a speedy alternative for large scale detection of PVY from dormant tubers. The method is therefore recommended for testing of PVY in potato tubers on a commercial scale in a diagnostic laboratory.Resumen Se desarrolló y optimizó un procedimiento de transcripción reversa de tiempo real de TaqMan (RT-PCR de tiempo real), y se comparó con otros métodos rutinarios para detectar al virus Y de la papa (PVY) en tubérculos de papa en dormancia. Se designaron tres pares de iniciadores y sondas y se evaluaron para su conveniencia para facilitar la detección de PVY por RT-PCR de tiempo real para todos los grupos de variantes, incluyendo PVY O , PVY N , PVY N:O (0PVY N-Wi ) y PVY NTN . Entre las combinaciones probadas de iniciadores y sondas, la combinación de PVY-1 produjo el valor del ciclo del umbral más bajo (Ct) con 25.75. El procedimiento se optimizó mas adelante mediante el ajuste de varios parámetros, incluyendo la concentración iniciador/ sonda, volumen de reacción, ciclos de amplificación, y muestras maestras de diferentes fuentes. Se empleó posteriormente el RT-PCR de tiempo real para detectar PVY de tubérculos en dormancia de diferentes variedades y campos de papa, y se compararon los resultados con los obtenidos por la RT-PCR convencional, la serología con enzimas conjugadas (ELISA), en pruebas de brotes y en crecimiento. De 1069 tubérculos probados infectados con un solo virus (PVY solamente), ambos formatos de RT-PCR detectaron P...

RT-PCR and Real-Time RT-PCR Methods for the Detection of Potato Virus Y in Potato Leaves and Tubers

“…3 . The relative performance of traditional two-step RT-PCR and real-time RT PCR protocols and how they compare to the more common ELISA assay has been studied and reported by Singh et al [ 5 ]. A detailed costing of reagents, equipment, and labor for each protocol is shown in Note 3 .…”

“…The mosaic symptoms of PVY are diffi cult to observe on many potato varieties, hampering accurate detection of the virus visually. Also, observation of symptoms can only be done on foliage, and ELISA can only be applied to foliage or tuber sprouts, requiring either fresh tissue during the growing season or signifi cant time and effort in sprouting or grow-out of dormant tubers after harvest [ 5 ]. In addition, there are many strains of PVY that vary in the type and severity of symptoms [ 7 ], and visual observation and ELISA methods are limited in their ability to differentiate them [ 6 ].…”

“…Unlike visual or ELISA screening, PCR methods can be used to rapidly screen tubers for PVY immediately after harvest [ 5 ]. PCR methods are also ideal for differentiating between different PVY strains, and can be multiplexed to detect multiple strains within the same sample or plant [ 8 ].…”

The Adaptation of Silica Capture RT-PCR for the Detection of Potato Virus Y

“…Detection of potato viruses, based on their protocol, was more sensitive compared to RNA isolated via RNAeasy kit (Singh et al 2002). Moreover it was shown to be beneficial for PVY detection by real time RT-PCR (Singh et al 2012). However, the protocol developed by Singh et al (2002) is composed of many steps, includes sample digestion with DNAse I and proteinase K as well as organic solvents for extraction and precipitation steps (Singh et al 2002).…”

“…Reverse transcription PCR is much more sensitive and since 1990s has been adopted for detection of potato viruses directly in tuber extracts (Spiegel and Martin 1993;Singh and Singh 1996;Singh 1998;Singh et al 2002;Crosslin and Hamlin 2011). Many variations of RT-PCR have been developed focusing on (i) identification and elimination of factors negatively affecting virus detection in tubers (Singh and Singh 1996), (ii) development of standardized protocols (Singh 1998;Crosslin and Hamlin 2011), (iii) simplification of sample preparation (Nolasco et al 1993;Singh et al 2004), (iv) multiplexing (Nie and Singh 2000;Singh and Nie 2003;Du et al 2006;Li et al 2008;) and (v) real time detection (Knorr et al 1996;Boonham et al 2000;Agindotan et al 2007;Mortimer-Jones et al 2009;Singh et al 2012). However, reports comparing performance of RT-PCR (Barker et al 1993;Bolotova et al 2009), and real-time RT-PCR (Fox et al 2005) with grow-out test have shown that the reliability of the latter method is either equal or more.…”