Biomedical subjects

N Boonham

Publications and source records attributed to N Boonham.

At least 19 recordsLinked to original sources

PubMed2006

Occurrence of two different types of RNA-5-containing beet necrotic yellow vein virus in the UK.

Two types of RNA-5-containing beet necrotic yellow vein virus (BNYVV) have been detected in the UK at different sites in Norfolk. On the basis of nucleotide (nt) sequence comparisons, one virus source (UK-MH) was clearly identified as P type BNYVV, a virus type that had previously only been detected in two widely separated parts of the world, France and Kazakhstan. The other virus source (UK-FF) has a complex genome composition. The analysed portions of its RNAs 2 and 4 are closely related to the corresponding portions in the RNAs of the East Asian A type isolate S, whereas those of its RNAs 1 and 3 resemble P type RNA 1 from Kazakhstan and European A type RNA 3, respectively. Interestingly, the P25 encoded on its RNA 3 has an unique TYHG tetrad in the highly variable amino acid positions 67-70. RNA 5 of the UK-FF BNYVV source shares properties with P type RNA 5, but also with East Asian types of RNA 5. The possible origin and epidemiology of BNYVV types is discussed.

Base Sequence↗

PubMed2006

Development of real-time and conventional RT-PCR assays for the detection of potato yellow vein virus (PYVV).

Potato yellow vein virus (PYVV) is considered a quarantine pathogen in the European and Mediterranean Plant Protection Organization (EPPO) area. This virus is widespread and damaging at its centre of origin in South America. Current detection methods are either time-consuming or difficult to interpret. This paper reports the development of a sensitive, high throughput, real-time reverse transcription (RT)-PCR assay, based on TaqMan chemistry, suitable for PYVV detection. In addition, a reliable conventional RT-PCR assay for PYVV detection is also presented. Although less sensitive (1000 times less sensitive in direct comparison), this method requires less sophisticated equipment and as such should be a useful alternative to the real-time technique in some testing laboratories. The two assays presented here could assist in the implementation of quarantine measures for PYVV identification and in routine indexing of PYVV for the production of virus-free seed potatoes in areas of South America where the virus is highly damaging.

Crinivirus↗

PubMed2006

Sweet problems: insect traits defining the limits to dietary sugar utilisation by the pea aphid, Acyrthosiphon pisum.

Plant phloem sap is an extreme diet for animals, partly because of its high and variable sugar content. The physiological and feeding traits of the pea aphid Acyrthosiphon pisum that define the upper and lower limits to the range of dietary sucrose concentrations utilised by this insect were determined principally using chemically defined diets containing 0.125-1.5 mol l(-1) sucrose. On the diets with 0.125 mol l(-1) and 1.5 mol l(-1) sucrose, the aphids died as larvae within 8 and 14 days of birth, respectively. On the other diets, 60-96% of aphids developed to adulthood, and the 0.5 mol l(-1) and 0.75 mol l(-1) diets supported the highest fecundity. The diet with 0.125 mol l(-1) sucrose was ingested at 36% of the rate of the 0.25 mol l(-1) sucrose diet, but >90% of ingested sucrose-carbon was assimilated on both diets. This suggests that the lower limit is dictated by the aphid feeding response, specifically, a requirement for a minimal concentration of sucrose for sustained feeding. The haemolymph osmotic pressure of aphids on diets with 0.125-1.5 mol l(-1) sucrose was up to 68% higher than on 0.125-1.0 mol l(-1) sucrose diets, but diet consumption and sucrose-carbon assimilation was not reduced on the very high sucrose diets relative to 1.0 mol l(-1) sucrose. This suggests that failure of the osmoregulatory capacity of the insects on high sucrose diets may define the upper limit to the range of dietary sucrose utilised by the aphids. The mean haemolymph osmotic pressure of aphids on plants with phloem sap containing 0.37-0.97 mol l(-1) sucrose was 1.61+/-0.063 MPa (mean +/- s.e.m.), not significantly different from that (1.47+/-0.059 MPa) on diets with 0.25-1.0 mol l(-1) sucrose. It is concluded that the osmoregulatory response of aphids to diets and plants are comparable, and, more generally, that the feeding and osmoregulatory capabilities of the aphids are compatible with the phloem sugar levels commonly encountered by aphids feeding on plants.

Animals↗

PubMed2005

A scientific note on the detection of honeybee viruses using real-time PCR (TaqMan) in Varroa mites collected from a Thai honeybee (Apis mellifera) apiary.

Bee parasitic mite syndrome is a disease complex of colonies simultaneously infested with Varroa destructor mites and infected with viruses and accompanied by high mortality. By using real-time PCR (TaqMan), five out of seven bee viruses were detected in mite samples (V. destructor) collected from Thailand. Moreover, the results of this study provide an evidence for the co-existence of several bee viruses in a single mite. This is also the first report of bee viruses in mites from Thailand.

Animals↗

PubMed2005

Investigating the specificity of real-time PCR assays using synthetic oligonucleotides.

Potato spindle tuber viroid (PSTVd) causes damaging diseases of solanaceous crops and is a quarantine pathogen in the European Union. Previously a one-tube real-time RT-PCR assay based on TaqMan chemistry was developed and shown to be ideally suited to PSTVd detection. However, since it was impossible to trace infected plant material for every published PSTVd sequence reported, in silico predictions were made about assay specificity based on the positions of nucleotide polymorphisms within the published viroid sequences and the regions of the primers and probe. The predictions could not be verified due to the absence of viroid material. This paper describes work investigating the detection of these sequence variants by designing synthetic oligonucleotides to sequences from the database and testing them with a real-time PCR assay. The results show that all PSTVd sequence variants are detected, and that the closely related Mexican papita viroid is also detected, although with a lower efficiency. The paper gives indications as to what effect nucleotide changes at different positions within primers and probes might do and should aid in the testing of future assays, although it is difficult to draw fixed rules about the possible effect changes may have.

DNA Primers↗

PubMed2005

The use of real-time RT-PCR (TaqMan) and post-ELISA virus release for the detection of Beet necrotic yellow vein virus types containing RNA 5 and its comparison with conventional RT-PCR.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Beet necrotic yellow vein virus (BNYVV). The two assays designed were a broad-spectrum one that detected RNA 2 from all types and a second designed to detect types containing RNA 5. The assays were validated against a range of different isolates from Europe and the Far East. These real-time assays were compared to a conventional RT-PCR assay for the detection of RNA 5. Sensitivity comparisons showed that for the detection of RNA 5, TaqMan was 10,000 times more sensitive than the conventional RT-PCR assay. Further improvements were made to the test procedure by using post-ELISA virus release (VR), as an alternative to RNA extraction. This significantly increased the speed of processing samples and reduced the staff input required, allowing the TaqMan assay to be used routinely as part of an annual survey of UK field samples.

Beta vulgaris↗

PubMed2005

On-site DNA extraction and real-time PCR for detection of Phytophthora ramorum in the field.

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.

DNA↗

PubMed2004

Development of a real-time RT-PCR assay for the detection of potato spindle tuber viroid.

Potato spindle tuber viroid (PSTVd) is a quarantine pathogen in the European Union and causes damaging diseases of solanaceous crops. Under the EU Plant Health directive 2000/29/EC, countries must have the ability to detect and identify accurately and rapidly the introduction of harmful organisms in plants or plant products; furthermore, if the quarantine pathogen is found, be able to survey extensively for it. In this respect, PSTVd poses an interesting technical problem, since its RNA does not code for any proteins and thus any diagnostic method must be based on the detection of the RNA and be suitable for scaling up to testing large sample numbers. With this in mind a one-tube real-time RT-PCR assay based on TaqMan chemistry was developed. Investigations were carried out into various aspects of the assay relevant to the efficient amplification of targets that have a significant amount of secondary structure such as viroids. Thus comparisons were made of reverse transcription temperature, concentration and type of reverse transcriptase, RNA denaturation, sample purity and single versus two-tube reaction format. The assay developed was shown to be able to detect a wide range of isolates of PSTVd and in comparison with a chemi-luminescent hybridisation system was shown to be 1000-fold more sensitive. A further significant advantage of this assay format compared with hybridisation is that it is suitable for scaling up to large sample numbers using robotic liquid handling systems.

DNA Primers↗

PubMed2003

Detection of potato viruses using microarray technology: towards a generic method for plant viral disease diagnosis.

Currently, most diagnostic methodology is geared towards detection of a very specific target species and often a number of assays need to be run in parallel to reach a result. The generic methods that are available for virus testing tends to give identification to the genus level only. The method described in this paper addresses this problem by exploiting a technology that has potential to test for a large number of targets in a single assay. Using the array constructed, the method was able to detect several common potato viruses (PVY, PVX, PVA, PVS) in single and mixed infections. The method was shown to be able to discriminate sequences with less than 80% sequence identity but was able to detect sequence variants with greater than 90% sequence identity. Thus the method should be useful for discriminating at the species level, but able to cope well with the intrinsic variability found within the genomes of RNA viruses. The sensitivity of the assay was found to be comparable with ELISA. The paper illustrates a significant step forward in the development of diagnostic methodologies by presenting for the first time a method that could theoretically be used not just for viruses, but for all the plant pathogens and pests that a modern diagnostic laboratory would want to test for, in a single completely generic and highly parallel format.

Base Sequence↗

PubMed2003

The detection of recombinant, tuber necrosing isolates of Potato virus Y (PVY(NTN)) using a three-primer PCR based in the coat protein gene.

A simple and reliable procedure for reverse transcription-polymerase chain reaction (RT-PCR) detection and strain differentiation of Potato virus Y (PVY) was developed. Three primers were designed within the coat protein (CP) and nuclear inclusion protein b (NIb) region, exploiting a single base polymorphism identified as being present in all the recombinant PVY(NTN) isolates published. Samples infected with PVY produce a single band of 569 bp, while isolates belonging to PVY(NTN) strain give an additional band of 334 bp. The technique was tested on a collection of well-characterised isolates of PVY from a range of strains and was found to detect all of the isolates reported as belonging to the PVY(NTN) strain. All of the isolates detected possess a recombination event within the coat protein. Further sequence analysis revealed that all the recombinant PVY(NTN) isolates reported thus far would be detected using this assay, whilst isolates thought to be PVY(NTN) that do not possess the coat protein recombination event would not be detected.

Base Sequence↗

PubMed2002

Improved detection of Sugarcane yellow leaf virus using a real-time fluorescent (TaqMan) RT-PCR assay.

Yellow leaf syndrome (YLS) of sugarcane has been associated with Sugarcane yellow leaf virus (ScYLV) and has been reported from most sugarcane growing countries around the world. As sugarcane is vegetatively propagated, it is important to use effective and sensitive detection methods to screen new propagating material. Virus detection in symptomatic tissue is currently achieved using enzyme linked immunosorbent assay (ELISA), tissue blot immunoassay (TBIA) or a conventional RT-PCR based assay. This paper reports the development of an improved assay based on multiplex real-time fluorescent RT-PCR. The new assay is 100-fold more sensitive than conventional RT-PCR, and incorporates a novel 'RNA specific' internal positive control (based around the intron of the caffeic acid 3-o-methyltransferase gene) to guard against false negative results. The paper also describes the comparison of eight RNA extraction methods for sugarcane tissue giving a number of alternatives for different laboratory situations. The sensitivity of this assay has allowed the detection of ScYLV in many samples that were thought to be healthy following conventional testing (RT-PCR, ELISA or TBIA). The detection of ScYLV using this TaqMan assay can be applied to the production of ScYLV-free plants and prevents its spread through the propagation material.

Luteovirus↗

PubMed2002

The detection of Tomato spotted wilt virus (TSWV) in individual thrips using real time fluorescent RT-PCR (TaqMan).

Tomato spotted wilt virus (TSWV) is an important virus, economically in the UK, causing damaging disease in ornamental and vegetable crops. The virus is vectored by several species of thrips, most importantly the western flower thrips (Frankliniella occidentalis Pergande [Thysanoptera: Thripidae]). The vector thrips themselves constitute a damaging pest and are difficult to control completely. Monitoring thrips numbers is an important part of the control of virus, but does not give information on how many of the thrips are viruliferous. Monitoring the presence of viruliferous thrips at an early stage of an epidemic may lead to improved disease control, since virus can be spread effectively whilst vector pressure is low and symptoms may take several weeks to appear on some hosts. This paper describes the development of a sensitive and robust, high-throughput method for the detection of TSWV in individual insects based on TaqMan chemistry. The method incorporates a novel RNA specific internal control to increase the reliability of the results. Results are also presented on comparisons of different extraction methods, including insects taken from sticky traps, for high-throughout testing. Implementation of a method such as this for the reliable detection of TSWV in individual thrips would aid the understanding of the progress of TSWV epidemics, and offer an early disease warning system for growers.

Animals↗

PubMed2001

Detection of different strains of Potato virus Y and their mixed infections using competitive fluorescent RT-PCR.

A competitive fluorescent RT-PCR assay (CF RT-PCR) was developed for the rapid and reliable detection and discrimination of the two most common strains of Potato virus Y (PVY) found in potato (necrotic and ordinary). The assay incorporates two strain specific primers labelled with fluorescent labels, used in conjunction with a universal PVY primer. The strain specific primers compete for the same annealing site which further increases specificity. Discrimination is conferred by the fluorescent labels; green PCR products for PVY(O) and red for PVY(N), whilst mixed infections are detected as orange PCR products without the need for staining agarose gels. The assay can be scaled up for the processing of 96 samples simultaneously, with the detection of PCR products directly using a fluorescent microtitre plate reader. The assay successfully discriminated between 20 isolates of PVY tested, and could be used for the direct detection of PVY in potato tubers.

Antibodies, Viral↗

PubMed2000

Improved detection of Beet necrotic yellow vein virus in a DAS ELISA by means of antibody single chain fragments (scFv) which were selected to protease-stable epitopes from phage display libraries.

The detection of Beet necrotic yellow vein virus (BNYVV) in stored sugar beets by means of monoclonal antibodies or antibody single chain fragments (scFv) often poses problems, because the immunodominant C-terminal epitope of the viral coat protein is readily lost due to proteolysis. Clones which produce scFv specific for protease-stable BNYVV epitopes were selected from two naive phage display libraries. Fusion proteins of the scFv with a human IgG kappa chain (expressed from the newly designed vector pCL) or with alkaline phosphatase,respectively, allow the ELISA detection of BNYVV even in stored sugar beets with a sensitivity which was comparable or often higher than that achieved with polyclonal antibodies.

Antibodies, Monoclonal↗

PubMed2000

Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay.

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.

Betaproteobacteria↗

PubMed2000

Virus strain discrimination using recombinant antibodies.

Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological facilities. Single chain Fv antibody fragments (scFv) have been selected from a synthetic phage-antibody library by affinity selection with purified Potato virus Y, ordinary strain (PVYO). The scFv selected was specific for PVY and detected 7 out of 9 isolates of PVYO whilst it did not detect 15 isolates from the closely related necrotic strains PVYN and PVYNTN. In ELISA the scFv could be used to detect virus at concentrations of 50 ng/ml in plant sap and was shown to have similar limits of detection as commercially available PVY monoclonal antibodies. These results highlight the potential of the technology for the selection of strain specific antibodies with an affinity and assay sensitivity similar to traditional monoclonal antibodies and their use in viral diagnostics.

Antibodies, Viral↗

PubMed1998

Strain specific recombinant antibodies to potato virus Y potyvirus.

Single chain Fv antibody fragments have been selected from a synthetic phage-antibody library following three and four rounds of affinity selection with purified potato virus Y, common strain (PVY(O)). The selected fragments were highly specific for PVY and detected seven out of nine isolates of PVY(O) whilst failing to detect three isolates of PVY(N) and 12 isolates of PVY(NTN). Nucleotide sequence of the scFv genes showed the variable heavy fragments belonged to the human VH4 family, whilst the variable light fragments belonged to the Vlambda1 family. The fragments were used in ELISA to detect virus at concentrations of 50 ng/ml in plant sap and in comparisons with commercially available PVY monoclonal antibodies were shown to have similar limits of detection. This is the first report of the selection of a scFv specific for a member of the potyviridae, and its use in detecting and differentiating strains of PVY in infected plant sap. The results highlight the potential of the technology for the selection of strain specific antibodies with an avidity equivalent to traditional monoclonal antibodies raised against viral pathogens and their use for viral diagnosis.

Amino Acid Sequence↗

PubMed1998

The characterization of a subgenomic RNA and in vitro translation products of oat chlorotic stunt virus.

Oat chlorotic stunt virus (OCSV) is a 35 nm icosahedral plant virus comprising of a single capsid protein with a Mr of 48.2 kDa and a 4.1 kb single stranded, positive sense genomic RNA. Northern blot analysis detected a single 3' terminal subgenomic RNA in extracts from infected plants, which was also found to be encapsidated. Virion RNA directs the synthesis of a 23 kDa polypeptide in a rabbit reticulocyte in vitro translation system. Primer extension analysis has been used to map the end of both the genomic and subgenomic RNA's, and has shown the genomic size to be 4115 nucleotides in length. The results have enabled a model for the genome expression to be proposed.

Animals↗