Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan®MGB probes

doi:10.1016/j.jviromet.2011.05.027

Journal of Virological Methods

Volume 176, Issues 1–2, September 2011, Pages 32-37

https://doi.org/10.1016/j.jviromet.2011.05.027 Get rights and content

Abstract

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan^®MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10³–10¹⁰ TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidentalis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

Highlights

► Development of real time RT-PCR for Tomato spotted wilt virus (TSWV). ► Estimation of the number of RNA copies of TSWV genomic segment M. ► Quantitation of TSWV in different host plants and the thrips vector. ► Quantitation of different TSWV genotypes in mixed infections.

Introduction

Tomato spotted wilt virus (TSWV), the type member of the genus Tospovirus of the family Bunyaviridae, is one of the most economically significant plant viruses causing damage to diverse ornamental and vegetable crops worldwide (Adkins, 2000). TSWV has a wide host range including more than 1000 plant species (Hanssen et al., 2010). It is transmitted in a persistent manner by several thrips species (Thysanoptera: Thripidae), with Frankliniella occidentalis (Pergande) being its main vector. The virus can only be acquired effectively by first instar larvae, and it can only be inoculated by second instar larvae and adult thrips, after a latent period during which TSWV reaches the salivary glands where it replicates (Wijkamp et al., 1993b). TSWV virions are quasi-spherical, measure 80–110 nm in diameter and are composed of an outer membrane envelope derived from the host, with two virus-coded glycoproteins (G_N and G_C) which are embedded and projected from the surface. Inside there are several copies of the RNA dependent RNA polymerase (RdRp) and nucleoproteins which encapsidate the genome. This consists of three single-stranded negative-sense or ambisense RNA segments: the L segment (∼8.9 kb) encodes in the negative sense a putative RNA-dependent RNA polymerase; the M segment (∼4.8 kb) encodes in the positive sense the plant cell-to-cell movement protein NSm, and in the negative sense the precursor of surface glycoproteins, G_N/G_C, involved in TSWV transmission by thrips; and the S segment (∼2.9 kb) encodes in the positive sense the silencing suppressor NSs, and in negative sense, the nucleoprotein N (Pappu et al., 2009).

Eradication or control of TSWV is a very difficult task due to its wide host range, its effective spread by thrips, and its great ability to evolve and adapt to new situations (Adkins, 2000, Pappu et al., 2009). Breeding for resistance has been proven to be the most effective method of control, although only the resistance conferred by two genes, Sw-5 in tomato (Solanum lycopersicum) and Tsw in pepper (Capsicum annuum), has been found to be efficient against a wide spectrum of TSWV isolates (Pappu et al., 2009). However, resistance-breaking TSWV isolates have been reported in several countries after a few years of using these resistant cultivars (Margaria et al., 2007, Lopez et al., 2011).

Specific and sensitive detection as well as accurate estimation of the virus titer in plant hosts and thrips vectors are necessary to study different aspects of virus biology and epidemiology, as well as to develop and evaluate strategies of disease control. Quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) has been widely used for diagnosis and quantitation of diverse human, animal and plant viruses (Mackay et al., 2002) because of its high sensitivity, specificity and reproducibility without the need for post-PCR sample processing. RT-qPCR has been developed to detect and quantify the genomic S segment of TSWV in plants (Roberts et al., 2000, Mortimer-Jones et al., 2009) and in thrips (Boonham et al., 2002), although it has only been assayed with genetically similar isolates without considering the worldwide genetic diversity of TSWV. Nucleotide analysis of a good number of worldwide TSWV isolates (Tsompana et al., 2005, Lopez et al., 2011) showed a relatively high genetic variability with two main genotypes (groups of isolates based on genetic similarity) named genotype A and genotype B. It would be valuable to develop a RT-qPCR valid for all TSWV isolates and/or able to discriminate these genotypes.

In this work, a RT-qPCR method for universal detection and quantitation of TSWV RNA of the genomic M segment was developed using primers and a TaqMan^®MGB probe based on conserved sequences of the TSWV M segment. Also, genotype-specific TaqMan^®MGB probes were designed to detect and monitor the accumulation of the two TSWV genotypes in single and mixed infections. These protocols were validated in different plant hosts and in thrips with different TSWV isolates in single and mix infections.

Section snippets

Virus isolates and biological assays

Twenty-six TSWV isolates, collected from tomato or pepper plants from Spain and the Brazilian isolate BR01 (kindly provided by Dr. Peters, Wageningen Agricultural University) were inoculated in plants of datura (Datura stramonium), tomato (cv. Robin, Royal Sluis, El Ejido, Almería, Spain) and/or pepper (cv. Dulce Italiano, Batlle, Molins de Rei, Barcelona, Spain) or were directly analyzed (Table 1). Plants were maintained in a chamber at a controlled temperature of 25 °C with a 14:10 h

Evaluation and comparison of TaqMan^®MGB probes for detection, discrimination and accumulation of TSWV isolates in different plant hosts and the thrips vector

To estimate the number of TSWV M segment RNA copies, three standard curves were prepared with probes P_U, P_A and P_B using 10-fold serial dilutions of the cognate RNA transcripts, containing 10¹⁰–10¹ copies. The three standard curves covered a wide dynamic range of eight log units of concentration and showed a strong linear relationship with a correlation coefficient greater than 0.990, as well as high amplification efficiency (>99.5%). The variation coefficient was very low (<1.0%). This

Discussion

Analysis of all available nucleotide sequences of TSWV M segment revealed two genotypes (groups of isolates), with nucleotide identities greater than 98% between isolates of the same genotype and about 93% between isolates of different genotypes (Tsompana et al., 2005, Lopez et al., 2011). RT-qPCR primers and TaqMan^®MGB probes were designed taking into account the worldwide genetic diversity of TSWV with the aim of obtaining a method valid for all TSWV isolates and able to differentiate the two

Acknowledgements

This work was supported by grants INIA RTA2008-00010-C03 and Generalitat Valenciana ACOMP/2009/103 and ACOMP/2010/085. D.E. Debreczeni is the recipient of a FPU predoctoral fellowship from MEC and B. Belliure was supported by a INIA-CCAA contract. We thank I. Ferriol, Dr. S. Ambrós and Dr. A. Olmos for technical advice; Dolores Comin for technical assistance; Dr. D. Peters and Dr. J. Contreras for kindly providing the TSWV isolate BR01 and a thrips colony, respectively; and Daniel R. Pearce for

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¹: Present address: Instituto de Biología Molecular y Celular de Plantas – Universidad Politécnica de Valencia (IBMCP, CSIC – UPV), 46022 Valencia, Spain.

²: Unidad Asociada IPAB-UA-CSIC-CIBIO, Universidad de Alicante, San Vicente del Raspeig, Alicante, Spain.

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Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan®MGB probes

Abstract

Highlights

Introduction

Section snippets

Virus isolates and biological assays

Evaluation and comparison of TaqMan®MGB probes for detection, discrimination and accumulation of TSWV isolates in different plant hosts and the thrips vector

Discussion

Acknowledgements

J. Virol. Methods

J. Virol. Methods

J. Virol. Methods

Virus Res.

J. Virol. Methods

J. Virol. Methods

J. Virol. Methods

Tomato spotted wilt virus-positive steps towards negative success

Mol. Plant Pathol.

Characterization of Tomato spotted wilt virus isolates that overcome the Sw-5 resistance gene in tomato and fitness assays

Phytopathol. Mediterr.

Emerging viral diseases of tomato crops

Mol. Plant Microbe Interact.

MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences

Brief. Bioinform.

3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures

Nucleic Acids Res.

Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan^®MGB probes

Evaluation and comparison of TaqMan^®MGB probes for detection, discrimination and accumulation of TSWV isolates in different plant hosts and the thrips vector