Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.

Development and comparison of a Primer-Probe Energy Transfer based assay and a 5 ' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus

In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5' conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2 x 10(8) to 2 x 10(2) copies/mu l. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5' conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV. (C) 2011 Elsevier B.V. All rights reserved.

Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus

The design and development of a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2 x 10(2) copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2 x 10(2) to 2 x 10(8) copies/mu l. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease. (C) 2010 Elsevier B.V. All rights reserved.

Genetic screening protocol for familial hypercholesterolemia which includes splicing defects gives an improved mutation detection rate

Familial hypercholesterolemia (FH) is a common single gene disorder, which predisposes to coronary artery disease. In a previous study, we have shown that in patients with definite FH around 20% had no identifiable gene defect after screening the entire exon coding area of the low density lipoprotein receptor (LDLR) and testing for the common Apolipoprotein B (ApoB) R3500Q mutation. In this study, we have extended the screen to additional families and have included the non-coding intron splice regions of the gene. In families with definite FH (tendon xanthoma present, n = 68) the improved genetic screening protocol increased the detection rate of mutations to 87%. This high detection rate greatly enhances the potential value of this test as part of a clinical screening program for FH. In contrast, the use of a limited screen in patients with possible FH (n = 130) resulted in a detection rate of 26%, but this is still of significant benefit in diagnosis of this genetic condition. We have also shown that 14% of LDLR defects are due to splice site mutations and that the most frequent splice mutation in our series (c.1845 + 11 c > g) is expressed at the RNA level. In addition, DNA samples from the patients in whom no LDLR or ApoB gene mutations were found, were sequenced for the NARC-1 gene. No mutations were identified which suggests that the role of NARC-1 in causing FH is minor. In a small proportion of families (

Isolation and detection of Campylobacter jejuni from chicken fecal samples by immunomagnetic separation–PCR

Campylobacter jejuni (C. jejuni) is one of the leading causes of bacterial food-borne disease worldwide. The presence of Campylobacter in chicken feces poses a high risk for contamination of chicken meat and for Campylobacter infections in human. Detection of this bacterium in chicken fecal specimens before slaughter is therefore vital to prevent disease transmission. By combining two techniques – immunomagnetic separation (IMS) and polymerase chain reaction (PCR), this study developed a reliable and specific method for rapid detection of C. jejuni in chicken fecal samples. The specificity of the assay was assured by two selection steps: 1) Dynabeads®M-270 Amine microbeads (2.8 µm in diameter) coated with C. jejuni monoclonal antibodies were used as the primary selection to isolate bacteria from fecal samples. 2) A PCR assay amplifying the Hippuricase gene was performed as the specific selection to accurately confirm the presence of C. jejuni. Without pre-enrichment, this method was able to detect approximately 10 CFU of C. jejuni in 1 µl of spiked feces within 3 h.

Molecular characterisation of Alternaria linicola and its detection in linseed

Nucleotide sequences of the ribosomal DNA (rDNA) internal transcribed spacers (ITS) 1 and 2 and a 1068 bp section of the beta-tubulin gene divided seven designated species of Alternaria into five taxa. Stemphylium botryosum formed a sixth closely related taxon. Isolates of A. linicola possessed an identical ITS sequence to one group of A. solani isolates, and two clusters of A. linicola isolates, revealed from beta-tubulin gene data to show minor variation, were as genetically similar to isolates of A. solani as they were to each other. We suggest, therefore, that A. linicola falls within the species A. solani. Similar results suggest that A. lini falls within the species A. alternata. RAPD analysis of the total genomic DNA from the Alternaria spp. concurred with the nucleotide sequence analyses. An oligonucleotide primer (ALP) was selected from the rDNA ITS1 region of A. linicola/A. solani. PCR with primers ALP and ITS4 (from a conserved region of the rDNA) amplified a c. 536 bp fragment from isolates of A. linicola and A. solani but not from other Alternaria spp. nor from other fungi which may be associated with linseed. These primers amplified an identical fragment, confirmed by Southern hybridization, from DNA released from infected linseed seed and leaf tissues. These primers have the potential to be used also for the detection of A. solani in host tissues.

Validation and application of a reporter gene assay for the determination of estrogenic endocrine disruptor activity in milk

Endocrine disruptors (EDs) are compounds known to interfere with the endocrine system by disturbing the action or pathways of natural hormones which may lead to infertility or cancer.Our diet is considered to be one of the main exposure routes to EDs. Since milk and dairy products are major components of our diet they should be monitored for ED contamination. Most assays developed to date utilise targeted, chromatography based methods which lack information on the biological activity and mixture effects of the monitored compounds.A biological reporter gene assay (RGA) was developed to assess the total estrogen hormonal load in milk. It has been validated according to EU decision 2002/657/EC. Analytes were extracted by liquid-liquid extraction with acetonitrile followed by clean up on a HLB column which yielded good recovery and small matrix effects. The method has been shown to be estrogen specific, repeatable and reproducible, with covariance values below 20%. In conclusion, this method enables the detection of low levels of estrogen hormonal activity in milk with a detection capability of 36pgg EEQ and has been successfully applied in testing a range of milk samples. © 2014 Elsevier Ltd.

Engineering plasmonic nanorod arrays for colon cancer marker detection

Engineering plasmonic nanomaterials or nanostructures towards ultrasensitive biosensing for disease markers or pathogens is of high importance. Here we demonstrate a systematic approach to tailor effective plasmonic nanorod arrays by combining both comprehensive numerical discrete dipole approximations (DDA) simulation and transmission spectroscopy experiments. The results indicate that 200×50 nm nanorod arrays with 300×500 nm period provide the highest FOM of 2.4 and a sensitivity of 310 nm/RIU. Furthermore, we demonstrate the use of nanorod arrays for the detection of single nucleotide polymorphism in codon 12 of the K-ras gene that are frequently occurring in early stages of colon cancer, with a sensitivity down to 10 nM in the presence of 100-fold higher concentration of the homozygous genotypes. Our work shows significant potential of nanorod arrays towards point-of-care applications in diagnosis and clinical studies.

Minimal residual disease detection in childhood acute lymphoblastic leukaemia patients at multiple time-points reveals high levels of concordance between molecular and immunophenotypic approaches

In this single centre study of childhood acute lymphoblastic leukaemia (ALL) patients treated on the Medical Research Council UKALL 97/99 protocols, it was determined that minimal residual disease (MRD) detected by real time quantitative polymerase chain reaction (RQ-PCR) and 3-colour flow cytometry (FC) displayed high levels of qualitative concordance when evaluated at multiple time-points during treatment (93.38%), and a combined use of both approaches allowed a multi time-point evaluation of MRD kinetics for 90% (53/59) of the initial cohort. At diagnosis, MRD markers with sensitivity of at least 0.01% were identified by RQ-PCR detection of fusion gene transcripts, IGH/TRG rearrangements, and FC. Using a combined RQ-PCR and FC approach, the evaluation of 367 follow-up BM samples revealed that the detection of MRD >1% at Day 15 (P = 0.04), >0.01% at the end of induction (P = 0.02), >0.01% at the end of consolidation (P = 0.01), >0.01% prior to the first delayed intensification (P = 0.01), and >0.1% prior to the second delayed intensification and continued maintenance (P = 0.001) were all associated with relapse and, based on early time-points (end of induction and consolidation) a significant log-rank trend (P = 0.0091) was noted between survival curves for patients stratified into high, intermediate and low-risk MRD groups.

Wilms' tumor gene 1 (WT1) expression in childhood acute lymphoblastic leukemia:a wide range of WT1 expression levels, its impact on prognosis and minimal residual disease monitoring

Wilms' tumor gene 1 (WT1) is overexpressed in the majority (70-90%) of acute leukemias and has been identified as an independent adverse prognostic factor, a convenient minimal residual disease (MRD) marker and potential therapeutic target in acute leukemia. We examined WT1 expression patterns in childhood acute lymphoblastic leukemia (ALL), where its clinical implication remains unclear. Using a real-time quantitative PCR designed according to Europe Against Cancer Program recommendations, we evaluated WT1 expression in 125 consecutively enrolled patients with childhood ALL (106 BCP-ALL, 19 T-ALL) and compared it with physiologic WT1 expression in normal and regenerating bone marrow (BM). In childhood B-cell precursor (BCP)-ALL, we detected a wide range of WT1 levels (5 logs) with a median WT1 expression close to that of normal BM. WT1 expression in childhood T-ALL was significantly higher than in BCP-ALL (P<0.001). Patients with MLL-AF4 translocation showed high WT1 overexpression (P<0.01) compared to patients with other or no chromosomal aberrations. Older children (> or =10 years) expressed higher WT1 levels than children under 10 years of age (P<0.001), while there was no difference in WT1 expression in patients with peripheral blood leukocyte count (WBC) > or =50 x 10(9)/l and lower. Analysis of relapsed cases (14/125) indicated that an abnormal increase or decrease in WT1 expression was associated with a significantly increased risk of relapse (P=0.0006), and this prognostic impact of WT1 was independent of other main risk factors (P=0.0012). In summary, our study suggests that WT1 expression in childhood ALL is very variable and much lower than in AML or adult ALL. WT1, thus, will not be a useful marker for MRD detection in childhood ALL, however, it does represent a potential independent risk factor in childhood ALL. Interestingly, a proportion of childhood ALL patients express WT1 at levels below the normal physiological BM WT1 expression, and this reduced WT1 expression appears to be associated with a higher risk of relapse.

Leukaemogenesis, gene interplay, and the role of the haemopoietic environment

Malignant initiation, leukaemic transformation, and disease progression in haematological malignancies involves a series of mutational events in genes involved in normal housekeeping functions of the cell. These acquired genetic changes can lead to either increased proliferation or a decreased rate of apoptosis, thus allowing expansion of the malignant clone. Although leukaemia can arise as a de novo disease, it has become increasingly clear that therapies, including the use of irradiation and/or chemotherapy, can give rise to malignancy. Therapy-associated myelodysplasia (t-MDS) and therapy-associated acute myeloid leukaemia (t-AML) account for 10-20% of new cases of these diseases. Although these secondary malignancies have been recognised as a clinical entity for nearly 30 years, molecular studies are now pinpointing various regions of the genome that are susceptible to DNA damage by these chemotherapeutic/radiotherapeutic strategies. The detection of new malignancies (both solid tumours and haematological tumours) following allogeneic bone marrow transplantation (BMT) is also providing us with some clues to the nature of leukaemogenesis, particularly with the observation that leukaemia can occur in donor cells postallogeneic BMT.

Meta-analysis of prostate cancer gene expression data identifies a novel discriminatory signature enriched for glycosylating enzymes

BACKGROUND: Tumorigenesis is characterised by changes in transcriptional control. Extensive transcript expression data have been acquired over the last decade and used to classify prostate cancers. Prostate cancer is, however, a heterogeneous multifocal cancer and this poses challenges in identifying robust transcript biomarkers.

METHODS: In this study, we have undertaken a meta-analysis of publicly available transcriptomic data spanning datasets and technologies from the last decade and encompassing laser capture microdissected and macrodissected sample sets.

RESULTS: We identified a 33 gene signature that can discriminate between benign tissue controls and localised prostate cancers irrespective of detection platform or dissection status. These genes were significantly overexpressed in localised prostate cancer versus benign tissue in at least three datasets within the Oncomine Compendium of Expression Array Data. In addition, they were also overexpressed in a recent exon-array dataset as well a prostate cancer RNA-seq dataset generated as part of the The Cancer Genomics Atlas (TCGA) initiative. Biologically, glycosylation was the single enriched process associated with this 33 gene signature, encompassing four glycosylating enzymes. We went on to evaluate the performance of this signature against three individual markers of prostate cancer, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) expression, prostate specific antigen (PSA) expression and androgen receptor (AR) expression in an additional independent dataset. Our signature had greater discriminatory power than these markers both for localised cancer and metastatic disease relative to benign tissue, or in the case of metastasis, also localised prostate cancer.

CONCLUSION: In conclusion, robust transcript biomarkers are present within datasets assembled over many years and cohorts and our study provides both examples and a strategy for refining and comparing datasets to obtain additional markers as more data are generated.

CGRP-cleavage enzyme detection using a biotinylated hydroxymate affinity probe

Introduction: Neuropeptides contribute to the pathophysiology of peripheral inflammation and a neurogenic component has been described for many inflammatory diseases, including periodontitis. Neuropeptides are susceptible to cleavage by peptidases and therefore the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies by our research group suggested that levels of the neuropeptide calcitonin gene-related peptide (CGRP) may be regulated by peptidases present in gingival crevicular fluid (GCF). Objectives: The aim of this work was to purify and partially characterize the GCF enzyme responsible for CGRP degradation using a biotinylated hydroxymate affinity probe (based on the P1-P4 amino acid sequence of the observed cleavage site) which we previously showed to inhibit CGRP degradation. Methods: Pooled healthy and pooled periodontitis GCF samples were subject to a pre-clear step with magnetic streptavadin beads. Healthy and diseased samples were incubated with the biotinylated hydroxymate probe (20 uM) after which biotinylated proteins were purified from the sample using magnetic streptavadin beads. Bound proteins were subjected to SDS-PAGE and western blotting. Biotin incorporated proteins were disclosed using a streptavadin horse radish peroxidase conjugate. Results: A band was disclosed in the periodontitis pooled sample at a molecular weight of approximately 60 kDa. The band was absent in the pooled healthy samples. As expected, when periodontitis samples were pre-boiled to denature proteins before the addition of the hydroxymate probe, no biotin incorporated band was present. Conclusions: This work demonstrates the purification and disclosure of a protein found specifically in periodontitis which binds to the specific biotinylated hydroxymate affinity probe based on the cleavage site of CGRP only when in its native form. We intend to scale up the sample size thus allowing the identification of the putative CGRP degrading peptidase using MALDI-mass spectrometry.
Funded by an IADR/GlaxoSmithKline Innovation in Oral Care Award

Application of molecular tools for detection of plant viruses

Grapevine leafroll disease (GLRD) is one of the most important virus diseases of grapevines worldwide, causing major economical impact. The disease has a complex aetiology and currently eleven phloem-limited viruses, termed in general Grapevine leafroll-associated virus (GLRaVs), have been identified. Two of the GLRaVs, GLRaV-1 and GLRaV-3, are included in the European certification scheme of propagation material. However, the flawed notion that GLRaV-3 is more frequent than GLRaV-1 and that all other GLRaVs are possibly not as relevant for GLRD, has until now precluded the development of specific serological and molecular detection assays and limited the scope of molecular characterization of the viruses known to be associated with the disease. Hence, few studies have addressed the phylodynamics of GLRaVs or even characterized the genetic structure of their natural populations. This generalized lack of molecular information, in turn underlie the deficient capacity to detect the viruses. The phylogenetic analyses were conducted on the basis of the heat shock protein 70 homologue (HSP70h) and the coat protein (CP) genes for GLRaV-1 and the HSP70h, the heat shock protein 90 homologue (HSP90h) and the CP genes for GLRaV-5. The data obtained for GLRaV-1 contributed 83 new CP sequences. This information was combined with previous analysis by other authors and used for the production of new polyclonal IgG, capable of detecting CP variants from all the phylogroups observed. Successful testing of this new tool included tissue print immunoblotting (TPIB) and in situ immunoassay (ISIA). The data obtained for GLRaV-5, contributed 61 new CP and 28 new HSP90h gene sequences. Eight phylogenetic groups were identified on the basis of the CP. Characterization of the genetic structure of the isolates revealed a higher diversity than previously reported and allowed the identification of dominant virus variants. For both GLRaV-1 and GLRaV-5, the effect of vegetative propagation on the virus transmission dynamics was addressed.

Mutations in the MECP2 Gene Are Not a Major Cause of Rett Syndrome-Like or Related Neurodevelopmental Phenotype in Male Patients

Rett syndrome is a genetic neurodevelopmental disorder that affects mainly girls, but mutations in the causative MECP2 gene have also been identified in boys with classic Rett syndrome and Rett syndrome-like phenotypes. We have studied a group of 28 boys with a neurodevelopmental disorder, 13 of which with a Rett syndrome-like phenotype; the patients had diverse clinical presentations that included perturbations of the autistic spectrum, microcephaly, mental retardation, manual stereotypies, and epilepsy. We analyzed the complete coding region of the MECP2 gene, including the detection of large rearrangements, and we did not detect any pathogenic mutations in the MECP2 gene in these patients, in whom the genetic basis of disease remained unidentified. Thus, additional genes should be screened in this group of patients.