Jenny Tomlinson

Advances in molecular phytodiagnostics - new solutions for old problems

In the last decade, developments in molecular (nucleic acid-based) diagnostic methods have made significant improvements in the detection of plant pathogens. By using methods such as the polymerase chain reaction (PCR), the range of targets that can now be reliably diagnosed has grown to the extent that there are now extremely few, known pathogens that cannot be identified accurately by using laboratory-based diagnostics. However, while the detection of pathogens in individual, infected samples is becoming simpler, there are still many scenarios that present a major challenge to diagnosticians and plant pathologists. Amongst these are the detection of pathogens in soil or viruses in their vectors, high throughput testing and the development of generic methods, that allow samples to be simultaneously screened for large numbers of pathogens. Another major challenge is to develop robust technologies that avoid the reliance on well-equipped central laboratories and making reliable diagnostics available to pathologists in the field or in less-developed countries. In recent years, much of the research carried out on phytodiagnostics has focussed in these areas and as a result many novel, routine diagnostic tests are becoming available. This has been possible due to the introduction of new molecular technologies such real-time PCR and microarrays. These advances have been complemented by the development of new nucleic acid extraction methods, increased automation, reliable internal controls, assay multiplexing and generic amplification methods. With developments in new hardware, field-portable real-time PCR is now also a reality and offers the prospect of ultra-rapid, on-site molecular diagnostics for the first time. In this paper, the development and implementation of new diagnostic methods based upon novel molecular techniques is presented, with specific examples given to demonstrate how these new methods can be used to overcome some long-standing problems.In the last decade, developments in molecular (nucleic acid-based) diagnostic methods have made significant improvements in the detection of plant pathogens. By using methods such as the polymerase chain reaction (PCR), the range of targets that can now be reliably diagnosed has grown to the extent that there are now extremely few, known pathogens that cannot be identified accurately by using laboratory-based diagnostics. However, while the detection of pathogens in individual, infected samples is becoming simpler, there are still many scenarios that present a major challenge to diagnosticians and plant pathologists. Amongst these are the detection of pathogens in soil or viruses in their vectors, high throughput testing and the development of generic methods, that allow samples to be simultaneously screened for large numbers of pathogens. Another major challenge is to develop robust technologies that avoid the reliance on well-equipped central laboratories and making reliable diagnostics available to pathologists in the field or in less-developed countries. In recent years, much of the research carried out on phytodiagnostics has focussed in these areas and as a result many novel, routine diagnostic tests are becoming available. This has been possible due to the introduction of new molecular technologies such real-time PCR and microarrays. These advances have been complemented by the development of new nucleic acid extraction methods, increased automation, reliable internal controls, assay multiplexing and generic amplification methods. With developments in new hardware, field-portable real-time PCR is now also a reality and offers the prospect of ultra-rapid, on-site molecular diagnostics for the first time. In this paper, the development and implementation of new diagnostic methods based upon novel molecular techniques is presented, with specific examples given to demonstrate how these new methods can be used to overcome some long-standing problems.

Methods in virus diagnostics: from ELISA to next generation sequencing.

Despite the seemingly continuous development of newer and ever more elaborate methods for detecting and identifying viruses, very few of these new methods get adopted for routine use in testing laboratories, often despite the many and varied claimed advantages they possess. To understand why the rate of uptake of new technologies is so low, requires a strong understanding of what makes a good routine diagnostic tool to begin. This can be done by looking at the two most successfully established plant virus detection methods: enzyme-linked immunosorbant assay (ELISA) and more recently introduced real-time polymerase chain reaction (PCR). By examining the characteristics of this pair of technologies, it becomes clear that they share many benefits, such as an industry standard format and high levels of repeatability and reproducibility. These combine to make methods that are accessible to testing labs, which are easy to establish and robust in their use, even with new and inexperienced users. Hence, to ensure the establishment of new techniques it is necessary to not only provide benefits not found with ELISA or real-time PCR, but also to provide a platform that is easy to establish and use. In plant virus diagnostics, recent developments can be clustered into three core areas: (1) techniques that can be performed in the field or resource poor locations (e.g., loop-mediated isothermal amplification LAMP); (2) multiplex methods that are able to detect many viruses in a single test (e.g., Luminex bead arrays); and (3) methods suited to virus discovery (e.g., next generation sequencing, NGS). Field based methods are not new, with Lateral Flow Devices (LFDs) for the detection being available for a number of years now. However, the widespread uptake of this technology remains poor. LAMP does offer significant advantages over LFDs, in terms of sensitivity and generic application, but still faces challenges in terms of establishment. It is likely that the main barrier to the uptake of field-based technologies is behavioural influences, rather than specific concerns about the performance of the technologies themselves. To overcome this, a new relationship will need to develop between centralised testing laboratories offering services and those requiring tests; a relationship which is currently in its infancy. Looking further into the future, virus discovery and multiplex methods seem to converge as NGS becomes ever cheaper, easier to perform and can provide high levels of multiplexing without the use of virus specific reagents. So ultimately the key challenge from a routine testing lab perspective will not be one of investment in platforms-which could even be outsourced to commercial sequencing services-but one of having the skills and expertise to analyse the large datasets generated and their subsequent interpretation. In conclusion, only time will tell which of the next-generation of methods currently in development will become the routine diagnostics of the future. This will be determined through a combination of factors. And while the technology itself will have to offer performance advantages over existing methods in order to supplant them, it is likely to be human factors e.g., the behaviours of end users, laboratories and policy makers, the availability of appropriate expertise, that ultimately determine which ones become established. Hence factors cannot be ignored and early engagement with diagnostic stakeholders is essential.

On-Site DNA Extraction and Real-Time PCR for Detection of Phytophthora ramorum in the Field

ABSTRACT Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.

Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field.

ABSTRACT Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.

Rapid Detection of Phytophthora ramorum and P. kernoviae by Two-Minute DNA Extraction Followed by Isothermal Amplification and Amplicon Detection by Generic Lateral Flow Device

ABSTRACT A method for nucleic-acid-based detection of pathogens in plant material has been developed which comprises a simple and rapid method for extracting DNA on the nitrocellulose membranes of lateral-flow devices, loop-mediated isothermal amplification (LAMP) of target DNA using labeled primers, and detection of the generically labeled amplification products by a sandwich immunoassay in a lateral-flow-device format. Each of these steps can be performed without specialist equipment and is suitable for on-site use, and a result can be obtained in just over an hour. A LAMP assay for the detection of plant DNA (cytochrome oxidase gene) can be used in conjunction with pathogen-specific assays to confirm negative results. The use of this method is demonstrated for the detection of Phytophthora ramorum, the causal agent of sudden oak death and dieback/leaf blight in a range of tree, shrub, and herbaceous species, and the recently described pathogen P. kernoviae.

Detection of Botrytis cinerea by loop-mediated isothermal amplification.

Aims:  To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting.

Exploiting generic platform technologies for the detection and identification of plant pathogens

The detection and identification of plant pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic skills to modern molecular-based methods. The wide range of methods employed reflects the great diversity of plant pathogens and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and techniques. Real-time polymerase chain reaction (PCR) is emerging as one such generic platform technology and one that is well suited to high-throughput detection of a limited number of known target pathogens. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal health, homeland security, biosecurity as well as plant health. Progress with developing generic techniques for plant pathogen identification, particularly of unknown samples, has been less rapid. Diagnostic microarrays and direct nucleic acid sequencing (de novo sequencing) both have potential as generic methods for the identification of unknown plant pathogens but are unlikely to be suitable as high-throughput detection techniques. This paper will review the application of generic technologies in the routine laboratory as well as highlighting some new techniques and the trend towards multi-disciplinary studies.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

SUMMARY The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the MspI satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus, up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

Development and inter-laboratory evaluation of real-time PCR assays for the detection of pospiviroids.

Assays based on real-time PCR (TaqMan) that can detect a number of viroids in the genus Pospiviroid have been developed and evaluated. The assays are designed for detecting viroids from tomato leaf material but detection from other solanaceous hosts of these viroids has been confirmed. These methods have been validated by nine laboratories and comprise a reliable set of assays for the detection of CEVd, TASVd, CLVd and a generic assay which will detect the six viroids of concern to European tomato growers: PSTVd, TCDVd, CEVd, CLVd, TASVd and CSVd.

Loop-mediated isothermal amplification for rapid detection of the causal agents of cassava brown streak disease

The causal agents of cassava brown streak disease have recently been identified as Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Primers have been developed for rapid detection of these viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP). Performance of the RT-LAMP assays compared favourably with published RT-PCR and real-time RT-PCR methods. Furthermore, amplification by RT-LAMP is completed in 40 min and does not require thermal cycling equipment. Modification of the RT-LAMP reactions to use labelled primers allowed rapid detection of amplification products using lateral flow devices containing antibodies specific to the incorporated labels, avoiding the need for fluorescence detection or gel electrophoresis.