Detection and quantification of Tomato mosaic virus in irrigation waters

Abstract

A quantitative RT real-time PCR method was developed for the detection and quantification of Tomato mosaic virus (ToMV) in irrigation waters. These have rarely been monitored for the presence of plant pathogenic viruses, mostly due to the lack of efficient and sensitive detection methods. The newly developed method presented here offers a novel approach in monitoring the health status of environmental waters. ToMV was reliably detected at as low as 12 viral particles per real-time PCR reaction, which corresponds to the initial concentration of approximately 4.2 · 10 -10 mg (6,300 viral particles) of ToMV per ml of sample. The sensitivity of the method was further improved by including the Convective Interaction Media Ò (CIM) monolithic chromatographic columns for quick and efficient concentration of original water samples. Seven out of nine water sources from different locations in Slovenia tested positive for ToMV, after concentrating the sample. Four samples tested ToMV-positive without the concentrating procedure. The presence and integrity of infective ToMV particles in the original sample, as well as in the chromatographic fraction, was confirmed using different methods from test plants, DAS ELISA to electron microscopy and real-time PCR. In this study, we propose a unique and simple diagnostic scheme for rapid, efficient, and sensitive monitoring of irrigation waters that could also be adopted for other plant, human or animal viruses.

Sensitive Detection of Multiple Rotavirus Genotypes with a Single Reverse Transcription-Real-Time Quantitative PCR Assay

“…The viral-RNA concentration associated with each cDNA dilution was recalculated based on the obtained regression equations and was used for calculating the coefficient of variance within each triplicate. cDNA dilutions whose triplicate C T values gave a coefficient of variance lower than 30% were assumed to be within the range of quantification (4,5). In regard to the range of detection, cDNA dilutions that gave a positive C T value in at least two of the triplicates were considered to be within the range of detection.…”

“…According to the nucleotide sequence of VP4 and VP7 genes, P and G genotypes are also distinguished. The most prevalent rotavirus genotypes in humans are G1P [8], G2P [4], G3P [8], G4P [8], and G9P [8] (13). In the last few years, the G12 genotype has apparently spread from Southeast Asia to all continents, similar to the worldwide emergence of G9 strains in the middle 1990s (22).…”

Concentration and detection of tobacco etch virus from irrigation water using real-time PCR

“…The largest obstacle in determining the infection route or behavior of a pathogenic virus is that the virus exists at an extremely low concentration in water. Although test methods utilizing conventional plaque assays or RT-PCR detection have been devised, it will remain very difficult to detect viruses in water (Boben, 2007). Consequently, more sensitive detecting methods combined with a procedure of concentration are necessary in studying WBPVs.…”

“…Recently, quantitative polymerase chain reaction (qPCR) methods have gained greater acceptance (Boben et al, 2007). Based on an internal control, qPCR can be divided into fluorescently labeled probes and fluorochromes (Lian et al, 2009;Luigi and Faggioli, 2011;Fedick et al, 2012;Gao et al, 2012).…”

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

“…These methods have proven successful in detecting and identifying different viruses on different hosts (Table 1). Since the viruses may be deactivated quickly when the infected plants dry up under field conditions, few studies have been done to detect the Table 1 plant viruses in soil and water (Boben et al 2007;Yang et al 2012). Viroids represent a group of extremely primitive pathogenic entities consisting exclusively of nucleic acids that are capable of independent replication and inducing diseases when introduced into susceptible plant cells (Narayanasamy 2011).…”