An evaluation of nucleic acid-based molecular methods for the detection of plant viruses: a systematic review.

Precise and timely diagnosis of plant viruses is a prerequisite for the implementation of efficient management strategies, considering factors like globalization of trade and climate change facilitating the spread of viruses that lead to agriculture yield losses of billions yearly worldwide. Symptomatic diagnosis alone may not be reliable due to the diverse symptoms and confusion with plant abiotic stresses. It is crucial to detect plant viruses accurately and reliably and do so with little time. A complete understanding of the various detection methods is necessary to achieve this. Enzyme-linked immunosorbent assay (ELISA), has become more popular as a method for detecting viruses but faces limitations such as antibody availability, cost, sample volume, and time. Advanced techniques like polymerase chain reaction (PCR) have surpassed ELISA with its various sensitive variants. Over the last decade, nucleic acid-based molecular methods have gained popularity and have quickly replaced other techniques, such as serological techniques for detecting plant viruses due to their specificity and accuracy. Hence, this review enables the reader to understand the strengths and weaknesses of each molecular technique starting with PCR and its variations, along with various isothermal amplification followed by DNA microarrays, and next-generation sequencing (NGS). As a result of the development of new technologies, NGS is becoming more and more accessible and cheaper, and it looks possible that this approach will replace others as a favoured approach for carrying out regular diagnosis. NGS is also becoming the method of choice for identifying novel viruses. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-024-00863-0.

Nanopore Technology Applied to Targeted Detection of Tomato Brown Rugose Fruit Virus Allows Sequencing of Related Viruses and the Diagnosis of Mixed Infections.

Tomato (Solanum lycopersicum) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.

Capacitive Field-Effect Biosensor Studying Adsorption of Tobacco Mosaic Virus Particles.

Plant virus-like particles, and in particular, tobacco mosaic virus (TMV) particles, are increasingly being used in nano- and biotechnology as well as for biochemical sensing purposes as nanoscaffolds for the high-density immobilization of receptor molecules. The sensitive parameters of TMV-assisted biosensors depend, among others, on the density of adsorbed TMV particles on the sensor surface, which is affected by both the adsorption conditions and surface properties of the sensor. In this work, Ta2O5-gate field-effect capacitive sensors have been applied for the label-free electrical detection of TMV adsorption. The impact of the TMV concentration on both the sensor signal and the density of TMV particles adsorbed onto the Ta2O5-gate surface has been studied systematically by means of field-effect and scanning electron microscopy methods. In addition, the surface density of TMV particles loaded under different incubation times has been investigated. Finally, the field-effect sensor also demonstrates the label-free detection of penicillinase immobilization as model bioreceptor on TMV particles.

Development of a colorimetric reverse transcription loop-mediated isothermal amplification assay for the detection of Mirafiori lettuce big-vein virus.

An RT-LAMP assay was developed to detect Mirafiori lettuce big vein virus (MiLBVV) and was compared with DAS-ELISA and RT-PCR. All primers were designed on the basis of the coat protein gene of the virus. In addition, a novel immunocapture (IC) RT-LAMP assay for rapid and easy detection of MiLBVV was developed, and factors such as safety, simplicity, cost, user-friendliness and safety were compared with those of DAS-ELISA, RT-PCR and RT-LAMP assays. Compared with DAS-ELISA and RT-PCR, RT-LAMP and IC-RT-LAMP had higher sensitivity (100-fold) but similar specificity, with the advantage of a shorter assay time and no need for RNA extraction (in IC-RT-LAMP). As RT-LAMP requires only very basic instruments and the results can be obtained by visual inspection (using GeneFinder™ dye), this technique provides a simple and reliable tool for laboratory research.

Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics.

BACKGROUND: Plant viruses in the genus Begomovirus, family Geminiviridae often cause substantial crop losses. These viruses have been emerging in many locations throughout the tropics and subtropics. Like many plant viruses, they are often not recognized by plant diagnostic clinics due in large part to the lack of rapid and cost effective assays. An isothermal amplification assay, Recombinase polymerase amplification (RPA), was evaluated for its ability to detect three begomoviruses and for its suitability for use in plant diagnostic clinics. Methods for DNA extraction and separation of amplicons from proteins used in the assay were modified and compared to RPA manufacturer's protocols. The modified RPA assays were compared to PCR assays for sensitivity, use in downstream applications, cost, and speed. RESULTS: Recombinase polymerase amplification (RPA) assays for the detection of Bean golden yellow mosaic virus, Tomato mottle virus and Tomato yellow leaf curl virus (TYLCV) were specific, only amplifying the target viruses in three different host species. RPA was able to detect the target virus when the template was in a crude extract generated using a simple inexpensive extraction method, while PCR was not. Separation of RPA-generated amplicons from DNA-binding proteins could be accomplished by several methods, all of which were faster and less expensive than that recommended by the manufacturer. Use of these modifications resulted in an RPA assay that was faster than PCR but with a similar reagent cost. This modified RPA was the more cost effective assay when labor is added to the cost since RPA can be performed much faster than PCR. RPA had a sensitivity approximate to that of ELISA when crude extract was used as template. RPA-generated amplicons could be used in downstream applications (TA cloning, digestion with a restriction endonuclease, direct sequencing) similar to PCR but unlike some other isothermal reactions. CONCLUSIONS: RPA could prove useful for the cost effective detection of plant viruses by plant diagnostic clinics. It can be performed in one hour or less with a reagent cost similar to that of PCR but with a lower labor cost, and with an acceptable level of sensitivity and specificity.

One-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of tomato torrado virus.

'Torrado' disease caused by tomato torrado virus (ToTV) is responsible for considerable losses in tomato production. Therefore, a one-step reverse transcription loop-mediated isothermal amplification protocol for early and fast detection of ToTV isolates has been developed. The RNA extracted from ToTV-infected plants was tested using this protocol with a set of six primers specific for the Vp35 coat protein gene sequence. The amplified products were analyzed using amplification curves, electrophoresis, and direct staining of DNA. The sensitivity of the protocol was tenfold higher than that of conventional RT-PCR. This new protocol is inexpensive, rapid, simple, and very sensitive.

Novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in South-East Australia.

A number of viruses from the genera Furovirus, Hordeivirus and Rymovirus are known to infect and damage the four major temperate cereal crops, wheat, barley, sorghum and oats. Currently, there is no active testing in Australia for any of these viruses, which pose a significant biosecurity threat to the phytosanitary status of Australia's grains industry. To address this, broad spectrum PCR assays were developed to target virus species within the genera Furovirus, Hordeivirus and Rymovirus. Five sets of novel genus-specific primers were designed and tested in reverse-transcription polymerase chain reaction assays against a range of virus isolates in plant virus diagnostic laboratories in both Australia and New Zealand. Three of these assays were then chosen to screen samples in a three-year survey of cereal crops in western Victoria, Australia. Of the 8900 cereal plants screened in the survey, all were tested free of furoviruses, hordeiviruses and rymoviruses. To date, there were no published genus-specific primers available for the detection of furoviruses, hordeiviruses and rymoviruses. This study shows for the first time a broad-spectrum molecular test being used in a survey for exotic grain viruses in Australia. Results from this survey provide important evidence of the use of this method to demonstrate the absence of these viruses in Victoria, Australia. The primer pairs reported here are expected to detect a wide range of virus species within the three genera.