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Radix spp. are intermediate host snails for digenean parasites of medical and veterinary importance. Within this genus, species differentiation using shell and internal organ morphology can result in erroneous species identification, causing problems when trying to understand the population biology of Radix. In the present study, DNA barcoding, using cox1 and ITS2 sequences, identified populations of Radix auricularia and Radix balthica from specimens originally morphologically identified as Radix peregra from the UK. Assessment of cox1 and ITS2 as species identification markers showed that, although both markers differentiated species, cox1 possessed greater molecular diversity and higher phylogenetic resolution. Cox1 also proved useful for gaining insights into the evolutionary relationships of Radix species populations. Phylogenetic analysis and haplotype networks of cox1 indicated that R. auricularia appeared to have invaded the UK several times; some haplotypes forming a distinct UK specific clade, whilst others are more akin to those found on mainland Europe. This was in contrast to relationships between R. balthica populations, which had low molecular diversity and no distinct UK specific haplotypes, suggesting recent and multiple invasions from mainland Europe. Molecular techniques therefore appear to be crucial for distinguishing Radix spp., particularly using cox1. This barcoding marker also enables the population biology of Radix spp. to be explored, and is invaluable for monitoring the epidemiology of fluke diseases especially in the light of emerging diseases and food security.

Background: High circulating tumor cell (CTC) counts are associated with poor prognosis in several cancers. Enrollment of patients on phase I oncology trials requires a careful assessment of the potential risks and benefits. Many patients enrolled on such trials using established eligibility criteria have a short life expectancy and are less likely to benefit from trial participation. We hypothesized that the incorporation of CTC counts might improve patient selection for phase I trials. Methods: This retrospective analysis evaluated patients who had baseline CTCs enumerated prior to their starting on a phase I trial. CTCs were enumerated using the CellSearch System. Results: Between January 2006 and December 2009 a total of 128 patients enrolled in phase I trials had CTC counts evaluated. Higher CTC counts as a continuous variable independently correlated with risk of death in this patient population (P = 0.006). A multivariate point-based risk model was generated using CTCs as a dichotomous variable (≥3 or 2 metastatic sites. Comparison of receiver operating characteristic curves demonstrated that the addition of baseline CTC counts improved the performance of the prospectively validated Royal Marsden Hospital phase I prognostic score, which now identifies three risk groups (P < 0.0001): good prognosis [score 0–1, median overall survival (OS) 63.7 weeks], intermediate prognosis (score 2–3, median OS 37.3 weeks), and poor prognosis (score 4, median OS 13.4 weeks). Conclusion: CTC enumeration improved the performance of a validated prognostic score to help select patients for phase I oncology trials. Clin Cancer Res; 17(15); 5188–96. ©2011 AACR.

A reverse transcription Linear-After-The-Exponential polymerase chain reaction (RT LATE-PCR) assay was evaluated for detection of foot-and-mouth disease virus (FMDV). This pan-serotypic assay targets highly conserved sequences within the 3D (RNA polymerase) region of the FMDV genome, and uses end-point hybridisation analysis of a single mismatch-tolerant low temperature probe to confirm the identity of the amplicons. An Armored RNA served as an internal control to validate virus negative results. The ability of the assay to identify FMDV was directly compared to a real-time RT-PCR assay routinely used by reference laboratories. The analytical sensitivity of the RT LATE-PCR assay was 10 genomic copies and the dynamic range of the test was identical to real-time RT-PCR based on decimal dilutions of an FMDV-positive sample. This pan-serotypic assay was able to detect FMDV in a broad range of clinical samples collected from field cases of FMD (n = 121), while samples of other viruses causing vesicular disease in livestock and genetic relatives of FMDV were negative. In addition to the laboratory-based utility of this diagnostic test, the RT LATE-PCR assay format has potential application for use in a portable ("point-of-care") device designed to achieve rapid detection of FMDV in the field.

This report describes the development of a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for the detection of swine vesicular disease virus (SVDV). The assay detects the virus rapidly, within 30-60 min and the result is visualised either by gel-electrophoresis or by the naked eye through the addition of SybrGreen. A collection of 28 SVDV isolates were tested positive, while heterologous viruses such as foot-and-mouth disease virus and vesicular stomatitis virus remained negative. The performance of the RT-LAMP was compared directly with real-time PCR using RNA from clinical samples including nasal swabs, serum and faeces. For nasal swabs and serum the sensitivity of the RT-LAMP was shown to be at least equivalent to real-time PCR. Interestingly, for faecal samples the RT-LAMP assay was shown to be even more sensitive than real-time PCR, possibly because it is less sensitive to inhibitory substances. This RT-LAMP assay provides a number of benefits for the diagnosis of SVD, since the assay is sensitive and rapid, and the isothermal amplification strategy used is not reliant upon expensive equipment it is particularly suited for "front line" diagnosis of SVD in modestly equipped laboratories, in field stations or in mobile diagnostic units.

This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot-and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants (Vigna unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and simple purification of RNA-2 containing capsids yields a preparation which is non-infectious.

The Atlantic salmon (Salmo salar) has been widely used as a model species in studies of olfactory signal transduction and processing. Here we report the isolation and characterisation of salmon olfactory receptor (SOR) and salmon vomeronasal receptor (SVR) partial sequences from Atlantic salmon. Six groups of SOR sequences (SORA–F) and three groups of SVR sequences (SVRA–C) were identified. All SORB, SORF, SVRB and SVRC sequences contained uninterrupted open reading frames. However, all SORA sequences and members of the SVRA sequence family contained multiple stop codons while SORC and SORE sequences were truncated in the 3′ region of the sequence. Full length SORF and almost complete SORB sequences displayed amino acid residues and motifs conserved in fish olfactory receptor genes. In sequence phylogenies, SOR sequences fell into the main olfactory receptor (MOR) type I clade and were most closely related to either δ or ζ reference sequences, while all SVR sequences grouped within a clade of fish type 2 vomeronasal receptor (V2R) sequences. A family of sequences (Sasa CaSR1–6), isolated using the same degenerate primers that amplified SVR sequences, clustered within a group of calcium sensing receptor (CaSR) sequences. Analysis of tissue expression patterns of sequences by reverse transcriptase polymerase chain reaction showed that they were transcribed in olfactory epithelium (SORB, SORF, all SVR and Sasa CaSR sequences), testis (SORB, SORD and Sasa CaSR) and/or anterior kidney (SORB and Sasa CaSR). Similar analysis of expression supported the identification of SORA sequences as non-transcribed pseudogene(s). Although the level of occurrence of OR pseudogenes is within the range found for other, well-characterised vertebrate OR genomes, it does not seem to reflect the importance of olfaction in the biology of the Atlantic salmon.

Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5-1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical 'hot-spot' mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.

Aims: A novel molecular assay for the detection of foot-and-mouth disease virus (FMDV) was developed using linear-after-the-exponential polymerase chain reaction (LATE-PCR). Methods and Results: Pilot experiments using synthetic DNA targets demonstrated the ability of LATE-PCR to quantify initial target concentration through endpoint detection. A two-step protocol involving reverse transcription (RT) followed by LATE-PCR was then used to confirm the ability of the assay to detect FMDV RNA. Finally, RT and LATE-PCR were combined in a one-step duplex assay for co-amplification of an FMDV RNA segment and an internal control comprised of an Armored RNA ® . In that form, each of the excess primers in the reaction mixture hybridize to their respective RNA targets during a short pre-incubation, then generate cDNA strands during a 3-min RT step at 60°C, and the resulting cDNA is amplified by LATE-PCR without intervening sample processing. Conclusions: The RT-LATE-PCR assay generates fluorescent signals at endpoint that are proportional to the starting number of RNA targets and can detect a range of sequence variants using a single mismatch-tolerant probe. Significance and Impact of the Study: In addition to offering improvements over current laboratory-based molecular diagnostic assays for FMDV, this new assay is compatible with a novel portable ('point-of-care') device, the BioSeeq ® II, designed for the rapid diagnosis of FMD in the field.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of African swine fever virus (ASFV). This assay targets the topoisomerase II gene of ASFV and its specificity was confirmed by restriction enzyme digestion of the reaction products. The analytical sensitivity of this ASFV LAMP assay was at least 330 genome copies, and the test was able to detect representative isolates of ASFV (n=38) without cross-reacting with classical swine fever virus. The performance of the LAMP assay was compared with other laboratory tests used for ASF diagnosis. Using blood and tissue samples collected from pigs experimentally infected with ASFV (Malawi isolate), there was good concordance between the LAMP assay and real-time PCR. In addition to detecting the reaction products using either agarose gels or real-time PCR machines, it was possible to visualise dual-labelled biotin and fluorescein ASFV LAMP amplicons using novel lateral flow devices. This assay and detection format represents the first step towards developing a practical, simple-to-use and inexpensive molecular assay format for ASF diagnosis in the field which is especially relevant to Africa where the disease is endemic in many countries.

DetectiV is a tool for analyzing pathogen-detection microarray datasets that allows simple visualisation, normalisation and significance testing., DNA microarrays offer the possibility of testing for the presence of thousands of micro-organisms in a single experiment. However, there is a lack of reliable bioinformatics tools for the analysis of such data. We have developed DetectiV, a package for the statistical software R. DetectiV offers powerful yet simple visualization, normalization and significance testing tools. We show that DetectiV performs better than previously published software on a large, publicly available dataset.

Heather Margaret Dukes went to Harare Central Hospital immediately after qualifying, developing skills in vascular access surgery that helped to start central Africa’s first renal unit. She did likewise in Coventry …

Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan assay, RT-LAMP appears to be sensitive, rapid, specific, and cost-effective, with the potential for field deployment and use by developing countries for FMDV surveillance.

RAPID and accurate diagnosis is necessary for the effective control of foot-and-mouth disease (FMD). Some laboratorybased methods are rapid, generating a result within a few hours, but the time taken to transport suspect clinical material to the laboratory can be lengthy, and this delay can preclude laboratory confirmation of disease. Notably, the implementation of the control strategy adopted by the UK government in the 2001 FMD epizootic – to slaughter animals on infected premises within 24 hours – meant that in the majority of cases, diagnosis based on clinical signs was not substantiated by laboratory investigation. Retrospective analyses have indicated that the reliance upon clinical diagnosis alone resulted in over-reporting of FMD, since the presence of FMD virus (FMDV) on 23 per cent of farms designated as infected premises was not confirmed by laboratory methods (Ferris and others 2006). These factors have given rise to the idea that the reliability of diagnosis may be improved by using rapid and sensitive tests that can be used in situ or close to a suspect premises, without transferring the samples to a central laboratory. The use of rapid diagnostic assays was recommended in two major reports considering the 2001 FMD outbreak (Anderson 2002, Royal Society 2002). Previous studies (Callahan and others 2002, Hearps and others 2002), have developed rapid reverse transcriptase-PCR (RT-PCR) assays for detecting FMDV; however, limitations in the hardware and some aspects of the protocols used have restricted the adoption of these assays for field detection of FMDV. In light of the general interest in this issue, this short communication describes recent progress that has been made towards the development of a mobile RT-PCR assay for FMDV detection. This study describes the evaluation of a portable real-time

Background: Circulating free DNA (cfDNA) represents a minimally-invasive resource for detecting mutations in advanced cancer patients. The primary objective of this study was to determine if cfDNA is representative of tumor tissue for multiplex mutation detection utilizing Sequenom MassARRAY. Methods: Metastatic colorectal, breast, melanoma and ovary cancer patients referred to the Drug Development Unit at the Royal Marsden Hospital between 9/09-8/10 gave their consent to provide DNA from matched archival formalin fixed paraffin embedded (FFPE) tumor and plasma. Samples mutation genotyping using the Sequenom MassARRAY technology and the OncoCartaTM Panel allowed the parallel analysis of 238 simple and complex mutations across 19 selected key oncogenes. Dilutions (10ng/µl to 0.01ng/µl) of HCT116 DNA containing KRAS G13D mutation were used to determine assay sensitivity and specificity. Results: Serial dilution spiking experiments revealed that the KRAS G13D mutation was reproducibly detectable to 40ng/ml of HCT116 DNA; 74 patient samples were then analyzed. The median quantity of cfDNA was 305ng/ml (range 10-32000). Of the 25 colorectal patients, the median quantity of cfDNA was 361ng/ml (range 189-4603); KRAS, BRAF and PIK3CA mutations were detected in 8 (31%), 3 (12%) and 3 (12%) tumor specimens respectively; concordant data for matched plasma tumor DNA was found in 88% of patients for KRAS, 100% for BRAF mutations and 33% for PIK3CA (1 patient had a PIK3CA mutation in primary and plasma; one patient had a mutation detected only in the primary; one patient had no mutation in the primary or plasma, but a mutation detected in a metastatic liver deposit). The recently reported oncogenic AKT1 E17K mutation was detected in 1 colorectal patient in tissue and plasma. Of the 19 breast cancer patients, the median cfDNA concentration was 354ng/ml (range 10-32000); PIK3CA H1047R mutation was detected in 4 (21%) FFPE tumor specimens; concordance between matched FFPE and cfDNA was 75% (3 out of 4 patients). The AKT1 E17K mutation was detected in 1 patient in both tissue and plasma. In 15 melanoma patients the median cfDNA concentration was 141ng/ml (range 46-1002); BRAF V600E, NRAS codon 61 and MET R970C mutations were detected in 4 (27%), 3 (20%) and 1 (7%) FFPE tumor specimens respectively. Concordance between matched FFPE and cfDNA was 50% for BRAF, 75% for NRAS and 100% for MET mutations. Another MET mutation, T992I, was found in plasma but not in tissue for one patient. Of the 15 ovary patients, the median cfDNA concentration was 261ng/ml (range 79-957). Two KRAS mutations and 1 PIK3CA H1047R mutation were detected in FFPE, but no mutation was found in any plasma sample. Conclusions: A high concordance in detected mutations was observed between FFPE tumor and matched cfDNA. cfDNA is representative of tumor DNA and may be used for the prospective selection of cancer patients for targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2011-3072

356 Background: Circulating free DNA (cfDNA) represents a minimally-invasive resource for detecting mutations in advanced cancer patients. The primary objective of this study was to determine if cfDNA is representative of tumor tissue for multiplex mutation detection utilizing Sequenom MassARRAY. Methods: Samples were spiked with dilutions (10ng/μl to 0.01ng/μl) of HCT116 DNA containing KRAS G13D mutations to determine assay sensitivity and specificity. Metastatic colorectal cancer patients referred to the Drug Development Unit at the Royal Marsden Hospital between 9/09-8/10 gave their consent to provide DNA from matched archival formalin fixed paraffin-embedded (FFPE) tumor and plasma. Samples had ∼200 described gene mutations genotyped using Sequenom MassARRAY (OncoCarta Panel). Results: Serial dilution spiking experiments revealed that the KRAS G13D mutation was reproducibly detectable to 40ng/mL of HCT116 DNA; 26 patient samples were then analyzed. KRAS, BRAF and PIK3CA mutations were detected in 8 (31%), 3 (12%) and 3 (12%) tumor specimens respectively; 100% concordance for KRAS status was observed between multiple FFPE biopsies from the same patient and analysis by Amplification Refractory Mutation System (ARMS)-Scorpion PCR. The median quantity of cfDNA was 353ng/ml (range 106-4603). Concordance between matched FFPE and cfDNA was 88% for KRAS and 100% for BRAF mutations. No patients with wildtype KRAS or BRAF tumor genotypes had mutations in their respective cfDNA confirming the high specificity of cfDNA analysis. Three patients had detectable PIK3CA mutations; 1 patient had a E346K mutation detected in both FFPE tissue and plasma; 1 patient had E545K detected only in FFPE and the other had E542K detected in a liver metastasis but not in the colorectal primary or plasma. The recently reported oncogenic AKT1 E17K mutation was detected in 1 patient in tissue and plasma. No mutations in any of the other tested oncogenes studied were detected. Conclusions: A high concordance in detected mutations was observed between FFPE tumor and matched cfDNA. cfDNA is representative of tumor DNA and may be used for the prospective selection of cancer patients for treatment with targeted therapeutics. No significant financial relationships to disclose.