Methods for identification of Staphylococcus aureus isolates in cases of bovine mastitis

A total of 272 staphylococcal isolates from cases of bovine mastitis (159 Staphylococcus aureus) belonging to 12 different species were identified with ID32 STAPH galleries, and 51 of them were confirmed by 16S rRNA gene (rrs) sequencing. The same isolates were examined for their hemolytic activity on sheep blood agar, DNase activity, and coagulase activity and with two rapid identification kits (Slidex Staph Plus kit and RAPIDEC Staph from Bio-Merieux). The results of this study confirm those obtained by other groups with hemolysis, DNase, and coagulase. Only 50% of S. aureus isolates from mastitis cases show coagulase activity after 4 h of incubation, and a 24-h incubation is necessary for the full sensitivity of this test. In contrast to results from other studies with human isolates, the Slidex Staph Plus kit was not sensitive enough for the identification of S. aureus from bovine mastitis samples. The aurease test of the RAPIDEC Staph kit showed 100% sensitivity and 100% specificity. Used in conjunction with hemolysis patterns, the RAPIDEC Staph kit is therefore very well adapted to rapid, efficient, and cost-effective identification of S. aureus in cultures from bovine mastitis samples. Sequencing of rrs genes also proved very efficient in identifying the Staphylococcus species encountered in these samples and confirming phenotypical identification results with unsatisfactory scores. With continuously improving technologies and decreasing costs, genetic identification methods like rrs gene sequencing will soon find a place in routine veterinary diagnostics.

SMART, a simple modular architecture research tool: Identification of signaling domains

Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.

A PCR method for the identification of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs

Aim: The aim of this study was to assess the discriminatory power and potential turn around time ( TAT) of a PCR-based method for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs. Methods: Screening swabs were examined using the current laboratory protocol of direct culture on mannitol salt agar supplemented with oxacillin (MSAO-direct). The PCR method involved pre-incubation in broth for 4 hours followed by a multiplex PCR with primers directed to mecA and nuc genes of MRSA. The reference standard was determined by pre-incubation in broth for 4 hours followed by culture on MSAO (MSAO-broth). Results: A total of 256 swabs was analysed. The rates of detection of MRSA using MSAO-direct, MSAO-broth and PCR were 10.2, 13.3 and 10.2%, respectively. For PCR, the sensitivity, specificity, positive predictive value and negative predictive values were 66.7% (95% CI 51.9 - 83.3%), 98.6% ( 95% CI 97.1 - 100%), 84.6% ( 95% CI 76.2 - 100%) and 95.2% ( 95% CI 92.4 - 98.0%), respectively, and these results were almost identical to those obtained from MSAO-direct. The agreement between MSAO-direct and PCR was 61.5% ( 95% CI 42.8 - 80.2%) for positive results, 95.6% ( 95% CI 93.0 - 98.2%) for negative results and overall was 92.2% ( 95% CI 88.9 - 95.5%). Conclusions: ( 1) The discriminatory power of PCR and MSAO-direct is similar but the level of agreement, especially for true positive results, is low. ( 2) The potential TAT for the PCR method provides a marked advantage over conventional methods. ( 3) Further modifications to the PCR method such as increased broth incubation time, use of selective broth and adaptation to real-time PCR may lead to improvement in sensitivity and TAT.

SeqDoC: rapid SNP and mutation detection by direct comparison of DNA sequence chromatograms

Background: This paper describes SeqDoC, a simple, web-based tool to carry out direct comparison of ABI sequence chromatograms. This allows the rapid identification of single nucleotide polymorphisms (SNPs) and point mutations without the need to install or learn more complicated analysis software. Results: SeqDoC produces a subtracted trace showing differences between a reference and test chromatogram, and is optimised to emphasise those characteristic of single base changes. It automatically aligns sequences, and produces straightforward graphical output. The use of direct comparison of the sequence chromatograms means that artefacts introduced by automatic base-calling software are avoided. Homozygous and heterozygous substitutions and insertion/deletion events are all readily identified. SeqDoC successfully highlights nucleotide changes missed by the Staden package 'tracediff' program. Conclusion: SeqDoC is ideal for small-scale SNP identification, for identification of changes in random mutagenesis screens, and for verification of PCR amplification fidelity. Differences are highlighted, not interpreted, allowing the investigator to make the ultimate decision on the nature of the change.

A concept for hip prosthesis identification using ultra wideband radar

Ultra wideband (UWB) radar has been extensively investigated both theoretically and practically for the identification buried artifacts. Ground probe radar (GPR) concentrates on the identification of lightly buried land mines, unexploded ordnance (UXO) and archeological targets. The same technology is proposed in a similar context for the rapid identification of in vivo implanted metallic prostheses. The technique is based on resonance based target identification and the paper investigates UWB scattering from a metallic hip prosthesis in free space as a first step in the identification process.

An application of near-infrared and mid-infrared spectroscopy to the study of natural halotrichites : halotrichite, apjohnite and wupatkiite

Near-infrared (NIR) and Fourier transform infrared (FTIR) spectroscopy have been used to determine the mineralogical character of isomorphic substitutions for Mg2+ by divalent transition metals Fe, Mn, Co and Ni in natural halotrichite series. The minerals are characterised by d-d transitions in NIR region 12000-7500 cm-1. NIR spectrum of halotrichite reveals broad feature from 12000 to 7500 cm-1 with a splitting of two bands resulting from ferrous ion transition 5T2g ® 5Eg. The presence of overtones of OH- fundamentals near 7000 cm-1 confirms molecular water in the mineral structure of the halotrichite series. The appearance of the most intense peak at around 5132 cm-1 is a common feature in the three minerals and is derived from combination of OH- vibrations of water molecules and 2 water bending modes. The influence of cations like Mg2+, Fe2+, Mn2+, Co2+, Ni2+ shows on the spectra of halotrichites. Especially wupatkiite-OH stretching vibrations in which bands are distorted conspicuously to low wave numbers at 3270, 2904 and 2454 cm-1. The observation of high frequency 2 mode in the infrared spectrum at 1640 cm-1 indicates coordination of water molecules is strongly hydrogen bonded in natural halotrichites. The splittings of bands in 3 and 4 (SO4)2- stretching regions may be attributed to the reduction of symmetry from Td to C2v for sulphate ion. This work has shown the usefulness of NIR spectroscopy for the rapid identification and classification of the halotrichite minerals.

Ambient ionisation mass spectrometry for the characterisation of polymers and polymer additives : A review

The purpose of this review is to showcase the present capabilities of ambient sampling and ionisation technologies for the analysis of polymers and polymer additives by mass spectrometry (MS) while simultaneously highlighting their advantages and limitations in a critical fashion. To qualify as an ambient ionisation technique, the method must be able to probe the surface of solid or liquid samples while operating in an open environment, allowing a variety of sample sizes, shapes, and substrate materials to be analysed. The main sections of this review will be guided by the underlying principle governing the desorption/extraction step of the analysis; liquid extraction, laser ablation, or thermal desorption, and the major component investigated, either the polymer itself or exogenous compounds (additives and contaminants) present within or on the polymer substrate. The review will conclude by summarising some of the challenges these technologies still face and possible directions that would further enhance the utility of ambient ionisation mass spectrometry as a tool for polymer analysis. (C) 2013 Elsevier B. V. All rights reserved.

Supplementary material : large scale read classification for next generation sequencing

Next Generation Sequencing (NGS) has revolutionised molecular biology, resulting in an explosion of data sets and an increasing role in clinical practice. Such applications necessarily require rapid identification of the organism as a prelude to annotation and further analysis. NGS data consist of a substantial number of short sequence reads, given context through downstream assembly and annotation, a process requiring reads consistent with the assumed species or species group. Highly accurate results have been obtained for restricted sets using SVM classifiers, but such methods are difficult to parallelise and success depends on careful attention to feature selection. This work examines the problem at very large scale, using a mix of synthetic and real data with a view to determining the overall structure of the problem and the effectiveness of parallel ensembles of simpler classifiers (principally random forests) in addressing the challenges of large scale genomics.

Characterization of ENU-induced mutations in red blood cell structural proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

Multiplexed tandem polymerase chain reaction identifies strong expression of oestrogen receptor and Her-2 from single, formalin-fixed, paraffin-embedded breast cancer sections

Aim To establish the suitability of multiplex tandem polymerase chain reaction (MT-PCR) for rapid identification of oestrogen receptor (ER) and Her-2 status using a single, formalin-fixed, paraffin-embedded (FFPE) breast tumour section. Methods Tissue sections from 29 breast tumours were analysed by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). RNA extracted from 10μm FFPE breast tumour sections from 24 of 29 tumours (14 ER positive and 5 Her-2 positive) was analysed by MT-PCR. After establishing a correlation between IHC and/or FISH and MT-PCR results, the ER/Her-2 status of a further 32 randomly selected, archival breast tumour specimens was established by MT-PCR in a blinded fashion, and compared to IHC/FISH results. Results MT-PCR levels of ER and Her-2 showed good concordance with IHC and FISH results. Furthermore, among the ER positive tumours, MT-PCR provided a quantitative score with a high dynamic range. Threshold values obtained from this data set applied to 32 archival tumour specimens showed that tumours strongly positive for ER and/or Her-2 expression were easily identified by MT-PCR. Conclusion MT-PCR can provide rapid, sensitive and cost-effective analysis of FFPE material and may prove useful as triage to identify patients suited to endocrine or trastuzumab (Herceptin) treatment.

Large scale read classification for next generation sequencing

Next Generation Sequencing (NGS) has revolutionised molecular biology, resulting in an explosion of data sets and an increasing role in clinical practice. Such applications necessarily require rapid identification of the organism as a prelude to annotation and further analysis. NGS data consist of a substantial number of short sequence reads, given context through downstream assembly and annotation, a process requiring reads consistent with the assumed species or species group. Highly accurate results have been obtained for restricted sets using SVM classifiers, but such methods are difficult to parallelise and success depends on careful attention to feature selection. This work examines the problem at very large scale, using a mix of synthetic and real data with a view to determining the overall structure of the problem and the effectiveness of parallel ensembles of simpler classifiers (principally random forests) in addressing the challenges of large scale genomics.

Cross-border malaria: a major obstacle for malaria elimination

Movement of malaria across international borders poses a major obstacle to achieving malaria elimination in the 34 countries that have committed to this goal. In border areas, malaria prevalence is often higher than in other areas due to lower access to health services, treatment-seeking behaviour of marginalised populations that typically inhabit border areas, difficulties in deploying prevention programs to hard-to-reach communities, often in difficult terrain, and constant movement of people across porous national boundaries. Malaria elimination in border areas will be challenging, and key to addressing the challenges is strengthening of surveillance activities for rapid identification of any importation or reintroduction of malaria. This could involve taking advantage of technological advances, such as spatial decision support systems, which can be deployed to assist program managers to carry out preventive and reactive measures, and mobile phone technology, which can be used to capture the movement of people in the border areas and likely sources of malaria importation. Additionally, joint collaboration in the prevention and control of cross-border malaria by neighbouring countries, and reinforcement of early diagnosis and prompt treatment are ways forward in addressing the problem of cross-border malaria.

Cloning and development of pathotype-specific SCAR marker associated with Sclerospora graminicola isolates from pearl millet

Downy mildew pathogen of pearl millet in India is associated with the spread of the highly virulent Sclerospora graminicola pathotype-1. Twenty-seven S. graminicola isolates were screened using 20 intersimple sequence repeats (ISSR). Dinucleotide repeat primer [17898A-(CA)(6) AC] amplified a similar to 600 bp fragment specific to five isolates of pathotype-1 (Sg 048, Sg 153, Sg 212, DM-11 and DM-90). The ISSR fragment linked with pathotype-1 was cloned successfully and sequenced. To convert ISSR fragments into pathotype-specific sequence characterised amplified region (SCAR) markers, PCR primers were designed using a sequence of the cloned DNA fragment. PCR amplification using SCAR primer pair (UOM3-Sg-Path1-F/R) amplified a single 284 bp band only in isolates of S. graminicola pathotype-1. This SCAR primer pair did not amplify the 284 bp product from the other five S. graminicola pathotypes or a negative control, which demonstrates primer specificity for pathotype-1. The SCAR primer pair (UOM3-Sg-Path1-F/R) obtained in this study will provide a valuable tool for rapid identification and specific detection of S. graminicola pathotype-1.

Materials selection in mechanical design for microsensors and microactuators

Microsensors and microactuators are vital organs of microelectromechanical systems (MEMS), forming the interfaces between controller and environment. They are usually used for devices ranging in size at sub-millimeter or micrometer level, transforming energy between two or more domains. Presently, most of the materials used in MEMS devices belong to the silicon material system, which is the basis of the integrated circuit industry. However, new techniques are being explored and developed, and the opportunities for MEMS materials selection are getting broader. The present paper tries to apply 'performance index' to select the material best suited to a given application, in the early stage of MEMS design. The selection is based on matching performance characteristics to the requirements. A series of performance indices are given to allow a wide range comparison of materials for several typical sensing and actuating structures, and a rapid identification of candidates for a given task. (C) 2002 Elsevier Science Ltd. All rights reserved.