Development of a portable, high throughput biosensor system for rapid plant virus detection

https://doi.org/10.1016/j.jviromet.2011.06.024Get rights and content

Abstract

Biosensors based on living cells are characterized by high sensitivity, selectivity and rapid response times. A novel portable cell biosensor system for the detection of plant viruses, based on immobilized ‘Vero’ cells carrying on their membrane virus specific antibodies was developed and was designated as High Throughput Bioelectric Recognition Assay (BERA-HTP). BERA-HTP was tested for the detection of purified Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Tobacco rattle virus (TRV) and of CMV and TRV in single, as well as in mixed infections in two different plant host species. The sensor was based on live, mammalian cells, the membrane of which has been artificially saturated with antibodies specific to different plant viruses. The attachment of PVY, CMV or TRV viral particles to the homologous electroinserted antibodies caused a virus-specific change of the cell membrane electric potential that was not observed with virus-free samples or with heterologous viruses. Fluorescence microscopy observations showed that attachment of virus particles to the cell membrane bearing the homologous antibody, was associated with a decrease of [Ca2+]cyt. The perspective for the development of BERA-HTP as a portable, reliable and rapid (duration of assay for 96 samples: ∼70 min) detection method of plant viruses in the field is discussed.

Highlights

► Cells as plant virus-specific biorecognition elements by engineering their membrane with respective antibodies. ► They were used in a novel portable cell biosensor system. ► The system was successfully used in specific plant virus detection. ► The assay was sensitive (1 ng ml−1), rapid and high throughput (duration of assay for 96 samples: ∼70 min).

Introduction

Biosensors based on living cells are known to have high sensitivity, selectivity, and rapid response times. These sensing systems have been employed in a wide range of environmental, chemistry and medical applications (Kintzios, 2007). Cell biosensors used for the detection of plant viruses are based on the change of the membrane electric potential of a host cell during its interaction with the corresponding viral particles. By applying this principle on immobilized plant protoplasts, a method called Bioelectric Recognition Assay (BERA) was presented, which allowed the detection of plant viral particles (Kintzios et al., 2001, Kintzios et al., 2004). Subsequently, a technique called Molecular Identification through “membrane-engineering” was developed, which involved the electroinsertion of virus-specific antibodies on the cell membrane. The attachment of a homologous virus triggered specific changes to the cell membrane electric potential. Based on this technique, Moschopoulou et al. (2008) and Perdikaris et al. (2009) developed a Vero-cell biosensor assay for the detection of Cucumber mosaic virus (CMV) and Hepatitis B Virus (HBV), respectively. Furthermore, the combination of the assay with specialized Artificial Neural Networks that were trained to recognize plant viruses according to biosensors’ responses has increased the performance of the BERA sensors (Frossyniotis et al., 2006).

In this work, a method optimized for high throughput detection of plant viruses and based on the BERA is presented. The new method, called BERA-HTP, uses “membrane-engineering” Vero cells which incorporate on their surface virus-specific antibodies at high density, rendering in this way each cell a very specific sensor of the interaction between a plant virus and the membrane-bound homologous antibodies. The method was tested for the identification of Cucumber mosaic virus (CMV), Tobacco rattle virus (TRV) and Potato virus Y (PVY) as purified particles as well as in sap-extracts derived from virus-infected plants. Most importantly, CMV and TRV-specific biosensors were tested for the detection of CMV and TRV in both single and mixed infections, in two different plant host species. These viruses possess a positive sense, single stranded RNA genome, belong to different virus genera and use different genome expression strategies. PVY (Potyvirus) infects species from the family Solanaceae, including potato, tobacco, pepper, tomato and solanaceous weeds. Found worldwide, PVY is the most damaging viral pathogen affecting potato (Valkonen, 2007) and is widespread in tomato crops causing serious yield losses (Aramburu et al., 2006, Rosner et al., 2000). CMV (Cucumovirus) has the widest host range among plant viruses causing substantial economic losses mainly in Solanaceae and Cucurbitaceae crops (Haase et al., 1989, Varveri and Boutsika, 1999). TRV (Tobravirus) infects experimentally a very wide range of plants and is an important pathogen of potato, tobacco and plant ornamentals. TRV isolates are characterized by a great variability in the nucleotide sequence of their coat protein gene resulting in inability of serological methods to detect in a single test all TRV serotypes (Robinson, 1989). Cucumber green mottle mosaic virus (CGMMV) (Tobamovirus) which has a narrow host range confined in Cucurbitaceae was used as an additional heterologous virus in CMV detection experiments. CGMMV is transmitted mechanically and through seed causing severe diseases primarily in cucumber and watermelon (Varveri et al., 2002).

The novel sensor is a portable multi-channel system, suitable for a high throughput assay (eight parallel tests in 5 min), which offers an optimal combination of high speed, sensitivity and low cost.

Section snippets

Sensor production

In order to construct the consumable part of the biosensor, membrane-engineered mammalian cells (see below) were prepared according to the procedure previously described by Moschopoulou et al. (2008). Briefly, Vero fibroblast cells were centrifuged at 100 × g for 6 min and then resuspended in Dulbecco's medium provided with 20% (v/v) fetal calf serum (FCS). Subsequently, cells were incubated together with the antibodies (0.5 μg ml−1) for 20 min on ice. Then, the cells–antibodies mixture was

DAS-ELISA

DAS-ELISA analysis with the appropriate virus antibodies confirmed that all plants were infected with the respective virus and virus combinations (data not shown). The detection sensitivity of DAS-ELISA, was determined using purified-virus, tenfold-diluted serially in PBS. DAS-ELISA detected purified CMV, PVY and TRV at concentrations as little as 100, 10 and 1 ng ml−1 PBS, respectively. In addition, PVY was detected in extracts from infected plants up to two times tenfold-diluted serially in sap

Discussion

Conventional methods in virus detection, such as RT-PCR and immunological assays, are used widely, despite their relatively long turnover times, as they offer high specificity, reproducibility and sensitivity. As an additional technological advance, the combination with robotic technology has enabled the analysis of an immense number of samples, nonetheless in expense of simplicity and of running cost (Lazcka et al., 2007). In certain applications such as phytosanitary control and

Conclusions

The present study is an important step towards the development of a portable plant virus detection system suitable for field applications and based on biosensor technology. Further experiments are required, possibly in combination with Artificial Neural Networks, in order to define the linear range of the response and to achieve a quantitative detection of plant viruses. Future tests using a wider range of plant viruses and their respective host species are necessary in order to confirm the

Acknowledgements

The authors acknowledge the contribution of Jan Kokkelink e.V. (The Hague, Netherlands) to manufacturing the consumable sensor-electrode interface. The research project was funded in part by the EMBIO Project of the Cypriot Ministry of Industry, Commerce and Tourism.

References (19)

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