1,064 research outputs found

    A method for automatically extracting infectious disease-related primers and probes from the literature

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    BACKGROUND: Primer and probe sequences are the main components of nucleic acid-based detection systems. Biologists use primers and probes for different tasks, some related to the diagnosis and prescription of infectious diseases. The biological literature is the main information source for empirically validated primer and probe sequences. Therefore, it is becoming increasingly important for researchers to navigate this important information. In this paper, we present a four-phase method for extracting and annotating primer/probe sequences from the literature. These phases are: (1) convert each document into a tree of paper sections, (2) detect the candidate sequences using a set of finite state machine-based recognizers, (3) refine problem sequences using a rule-based expert system, and (4) annotate the extracted sequences with their related organism/gene information. RESULTS: We tested our approach using a test set composed of 297 manuscripts. The extracted sequences and their organism/gene annotations were manually evaluated by a panel of molecular biologists. The results of the evaluation show that our approach is suitable for automatically extracting DNA sequences, achieving precision/recall rates of 97.98% and 95.77%, respectively. In addition, 76.66% of the detected sequences were correctly annotated with their organism name. The system also provided correct gene-related information for 46.18% of the sequences assigned a correct organism name. CONCLUSIONS: We believe that the proposed method can facilitate routine tasks for biomedical researchers using molecular methods to diagnose and prescribe different infectious diseases. In addition, the proposed method can be expanded to detect and extract other biological sequences from the literature. The extracted information can also be used to readily update available primer/probe databases or to create new databases from scratch.The present work has been funded, in part, by the European Commission through the ACGT integrated project (FP6-2005-IST-026996) and the ACTION-Grid support action (FP7-ICT-2007-2-224176), the Spanish Ministry of Science and Innovation through the OntoMineBase project (ref. TSI2006-13021-C02-01), the ImGraSec project (ref. TIN2007-61768), FIS/AES PS09/00069 and COMBIOMED-RETICS, and the Comunidad de Madrid, Spain.S

    Molecular detection of noroviruses in ready-to-eat foods and fruit products

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    In this PhD, three main goals were defined. The first goal consisted of the development and evaluation of a methodology for detection of noroviruses (NoV) in ready-to-eat (RTE) foods and soft red fruits while the second main goal included the evaluation of the murine norovirus 1 (MNV-1) as control reagent for different steps throughout the NoV detection protocols. Finally, a screening study on a selection of fruit produce products towards NoV presence was the third main goal of this PhD. To illustrate these goals, a literature study was performed in chapter 1. In this literature study, a brief overview of the most important food borne viruses was followed by a more detailed description of NoV in terms of classification, virion and genome structure. The NoV genotype most commonly identified in NoV gastroenteritis outbreaks (NoV GII.4) was described as well. The importance of NoV as a food borne pathogen was highlighted by data originating from official bodies such as CDC (Centers for Disease Control and Prevention; USA) and EFSA (European Food Safety Authority; Europe), accompanied with data gathered on own initiatives by research groups. The two main transmission routes of NoV contamination of foods (pre-harvest contamination via contact with contaminated water and (post-) harvest contamination via an infected food handler/picker) were investigated by summarizing and analyzing 59 NoV food borne outbreaks described between 2000 and 2010. Furthermore, the three main steps of NoV detection in food were portrayed in detail to prepare the development and evaluation of the NoV detection methodology in chapters 2, 3, 4 and 5. Finally, the use of adequate positive and negative controls to assure reliable detection of NoV in foods was illustrated. For the first and second goals of this PhD, a molecular assay for detection of the purified NoV genomic material was optimized and subsequently combined with protocols for extraction of (genomic material of) NoV from RTE foods and soft red fruits in chapters 4 and 5. MNV-1 was included in these protocols as control reagent. The molecular detection assay described in chapter 2 was a quantitative two-step multiplex real-time reverse transcriptase (RT-) PCR assay for simultaneous detection of NoV genogroup I (GI) and II (GII) and the murine norovirus 1 (MNV-1), the latter used as internal amplification control (IAC). For this multiplex assay, NoV GI and GII specific primers and hydrolysis probes designed by the European Committee for Standardization/ Technical Committee 275 / Working Group 6 /Task Group 4 on virus detection in foods (CEN/TC275/WG6/TAG4 working group) were combined with primers for murine norovirus 1 designed by Baert and colleagues (2008b). Evaluation of this multiplex assay showed a high concordance between the multiplex assay and the corresponding singleplex PCR assays. Specificity analysis of the multiplex assay by testing a NoV RNA reference panel and clinical GI and GII NoV samples showed that specific amplification of NoV GI and GII was possible. In addition, no cross-amplification was observed when subjecting a collection of bovine NoV and other (non-NoV) enteric viruses to the multiplex assay. Finally, MNV-1 was successfully integrated as IAC, although a sufficiently low concentration was needed to avoid interference with the possibility of the developed multiplex assay to quantitatively and simultaneously detect the presence of GI and GII NoV within one sample. During development of the multiplex real-time RT-PCR assay, contamination issues were encountered and the investigation towards the source of the positive no template controls (NTCs) was described in chapter 3. This investigation was believed to be necessary because of the need for reliable detection of 10 or less NoV genomic copies per PCR reaction, due to the low infectious dose of GI and GII NoV. In this chapter, a suspicion of well-to-well migration of positive control DNA (a short synthetic single stranded DNA (ssDNA) fragment) during real-time PCR runs was uttered as hypothetic cause of the positive NTCs. Results in this chapter showed that evaporation of water occurred during real-time PCR runs regardless of the DNA type, the reaction plate seal type and the use of mineral oil as cover layer. It was also suggested that co-evaporation of DNA took place, with an apparent negative correlation between the size of the DNA type and the extent of this co-evaporation. The use of mineral oil as cover layer and plasmid DNA as quantitative positive PCR control resulted in a complete absence of positive NTCs while only negligible effects were noticed on the performance of the real-time PCR. After development of the multiplex real-time RT-PCR assay and the resolving of the contamination issues, two protocols for extraction of (genomic material) of NoV from foods were evaluated towards robustness and sensitivity while MNV-1 was evaluated as process control in both protocols. The evaluation of a direct RNA extraction protocol for extraction of NoV genomic material (RNA) from RTE foods was described in chapter 4, while the evaluation of an elution-concentration protocol for extraction of NoV from soft red fruits was illustrated in chapter 5. For the RTE foods, the direct RNA extraction protocol made use of a guanidine isothiocyanate containing reagent to extract viral RNA from the food sample (basic protocol called TriShort), followed by an eventual concentration step using organic solvents (extended protocol called TriConc). The protocol for extraction of NoV from soft red fruits consisted of alkaline elution of NoV particles from the food, followed by polyethylene glycol (PEG) precipitation and organic solvent purification. For both protocols the RNA was subsequently purified. This purified RNA was detected by the multiplex real-time RT-PCR assay as described in chapter 2. To evaluate both NoV extraction methods towards sensitivity and robustness, the influence of (1) the NoV inoculum level and (2) different food types on the recovery of NoV from these foods was investigated. First of all, a significant influence of the NoV inoculum level on the recovery of NoV from foods was demonstrated for both protocols. High level inocula could be recovered from penne salad, selected as typical RTE food, with higher recovery success rates compared to low level inocula. For these high inoculum levels, the TriShort and TriConc protocols resulted in mean recovery efficiencies of >1 % and 0.1 to 10 %, respectively. Recovery of these low and high level NoV inocula from frozen raspberry crumb was possible with high recovery success rates and with mean recovery efficiencies of 10 to 30 % in most cases. Secondly, a significant influence of the food type on the recovery of NoV could be shown for both protocols. For the direct RNA extraction protocol, the TriConc protocol provided better NoV recoveries for soups, while TriShort and TriConc protocols performed likewise for composite meals and deli sandwiches, although NoV recovery from the latter food type was problematic. For the elution-concentration protocol, a significant influence of the soft red fruit product type on the recovery efficiency of NoV GI and MNV-1 was noticeable, while no significant differences could be shown for GII NoV. In general, the recovery of NoV was more efficient and successful from the strawberry puree compared to a frozen forest fruit mix and fresh raspberries. Regarding the evaluation of MNV-1 as control reagent, results from chapter 4 and chapter 5 suggested that a sufficient high concentration of the MNV-1 PC was needed to allow an estimation of possible inhibition of the RT-PCR or of inefficient virus extraction. When used as reverse transcription control or internal amplification control, the concentration should be adjusted to avoid interference with the quantitative properties of the developed multiplex real-time RT-PCR assay. Chapter 6 described the screening of 75 fruit products (raspberries, strawberries, cherry tomatoes and fruit salads) for NoV presence using the virus extraction protocol described in chapter 5 combined with the multiplex real-time RT-PCR assay illustrated in chapter 2. In total, 18 samples tested positive for GI and/or GII NoV genomic material despite a good bacteriological quality. The level of detected NoV genomic copies concentrations ranged between 2.5 and 5.0 logs per 10 grams of fruit sample. NoV GI and/or GII were found in 4/10, 7/30, 6/20 and 1/15 of the tested raspberries, cherry tomatoes, strawberries and fruit salad samples, respectively. However, confirmation of the positive real-time PCR results by sequencing genotyping regions in the NoV genome was not possible. The question whether or not these unexpected high number of NoV positive results obtained should be perceived as a public health threat was raised and discussed. In conclusion, methods for detection of NoV in RTE foods and soft red fruits were developed and evaluated towards sensitivity and robustness. For detection of NoV in soft red fruits and ready-to-eat foods, an elution-precipitation protocol and a direct RNA extraction protocol were combined with an optimized multiplex real-time RT-PCR assay leading to NoV detection protocols with detection limits of ~104 genomic copies / 10g food product. Influence of the NoV inoculum level and food type on NoV recovery was shown. Additionally, MNV-1 was successfully evaluated as control reagent, and suggestions were made towards its use. However, application of the method for NoV detection in fruit products has shown that interpretation of NoV presence by molecular methods is not straightforward and raises several questions, especially towards the public health safety

    Annotating genes and genomes with DNA sequences extracted from biomedical articles

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    Motivation: Increasing rates of publication and DNA sequencing make the problem of finding relevant articles for a particular gene or genomic region more challenging than ever. Existing text-mining approaches focus on finding gene names or identifiers in English text. These are often not unique and do not identify the exact genomic location of a study

    Development of Electronic Microarray Assays for the Detection of High Consequence Swine Viruses

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    This thesis describes the development and optimization of a seven-plex reverse transcription polymerase chain reaction (RT-PCR) and corresponding user-friendly electronic microarray for the detection of seven swine viruses: foot-and-mouth disease, swine vesicular disease, classical swine fever, vesicular exanthema of swine, African swine fever, porcine circovirus type 2 and porcine reproductive and respiratory syndrome. A panel of 58 strains of the viruses were successfully amplified and detected specifically on the NanoChip 400 microarray system while having no detection of 22 non-specific clinical material and non-target viruses and bacteria. Target viruses were also detected from clinical and biological materials spiked with viruses as early as 1 days post-infection. Detection limits ranged from 10 to 1000 copies for the targets. The assay was successfully transferred to the prototype Nexogen MDx portable and integrated system, where the fully automated system, with no manual handling from sample to detection, detected five of the seven viruses

    e-MIR2: a public online inventory of medical informatics resources

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    Background. Over the last years, the number of available informatics resources in medicine has grown exponentially. While specific inventories of such resources have already begun to be developed for Bioinformatics (BI), comparable inventories are as yet not available for Medical Informatics (MI) field, so that locating and accessing them currently remains a hard and time-consuming task. Description. We have created a repository of MI resources from the scientific literature, providing free access to its contents through a web-based service. Relevant information describing the resources is automatically extracted from manuscripts published in top-ranked MI journals. We used a pattern matching approach to detect the resources? names and their main features. Detected resources are classified according to three different criteria: functionality, resource type and domain. To facilitate these tasks, we have built three different taxonomies by following a novel approach based on folksonomies and social tagging. We adopted the terminology most frequently used by MI researchers in their publications to create the concepts and hierarchical relationships belonging to the taxonomies. The classification algorithm identifies the categories associated to resources and annotates them accordingly. The database is then populated with this data after manual curation and validation. Conclusions. We have created an online repository of MI resources to assist researchers in locating and accessing the most suitable resources to perform specific tasks. The database contained 282 resources at the time of writing. We are continuing to expand the number of available resources by taking into account further publications as well as suggestions from users and resource developers

    Fully integrated microsystem for bacterial genotyping

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    Methods for bacterial detection and identification has garnered renewed interest in recent years due to the infections they may cause and the antimicrobial resistances they can develop, the potential for bioterrorism threats and possible contamination of food/water supplies. Therefore, the rapid, specific and accurate detection of pathogens is crucial for the prevention of pathogen-related disease outbreaks and facilitating disease management as well as the containment of suspected contaminated food and/or water supplies. In this dissertation an integrated modular-based microfluidic system composed of a fluidic cartridge and a control instrument has been developed for bacterial pathogen detection. The integrated system can directly carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and can detect sequence variations in selected genes to allow for the identification of the bacterial species and even its strain. The unique aspect of this fluidic cartridge is its modular format with a task-specific module interconnected to a fluidic motherboard to permit the selection of a material appropriate for the given processing step(s). In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the fluidic cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and assembled into a small footprint instrument. The fluidic cartridge was capable of performing cell lysis, solidphase extraction of genomic DNA from the whole cell lysate, continuous flow PCR amplification of specific gene fragments, continuous flow ligase detection reaction to discriminate sequence variations and universal DNA array readout, which consisted of DNA probes patterned onto a planar polymer waveguide for evanescent excitation. The performance of the fluidic system was demonstrated through its successful application to the genetic detection of bacterial pathogens, such as Escherichia coli O157:H7, Salmonella, methicillin-resistant Staphylococcus aureus and multi-drug resistant Mycobacterium tuberculosis, which are major threats for global heath. The modular system, which could successfully identify several strains of bacteria in \u3c40 min with minimal human intervention and also perform strain identification, represents a significant contribution to pathogen detection

    Quantitation of Group A Rotavirus by real time Polymerase Chain Reaction in Children with varying severity of gastroenteritis

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    INTRODUCTION: Rotaviral gastroenteritis is one of the leading causes of mortality and morbidity in Indian children below 5 years of age. India spends about 2 to 3.4 billion rupees annually on the treatment of these cases. One in approximately every 260 children below five years of age die of rotavirus diarrhea every year. Faeco oral route is the most important mode of transmission and hence improvement in sanitation and general hygiene should ideally prevent transmission. However, even in the developed countries, this could not be achieved. It is difficult to prevent feco-orally transmitted diseases in developing countries without multiple preventive measures, including vaccination. The World Health Organization (WHO) has recommended incorporation of vaccines against rotavirus diarrhea in the national immunization schedules of developing countries with high diarrheal mortality. Though vaccines are available, they have to be in a reasonable price range to be affordable for the poorer countries which have the majority of the mortality and severe disease burden. To cater to this need in India, various indigenous vaccines are being developed. Rotavirus is a double stranded RNA virus belonging to the family Reoviridae. The genome is segmented. The virus is a triple layered particle with outer, middle and inner layers. Different serogroups are identified based on the antigenic specificity of the middle layer of the virus and electrophoretic mobility of the 11 RNA segments of the viral genome. Out of the seven different serogroups (A – G) identified, Groups A, B and C are associated with humans and group A viruses are most commonly associated with severe life threatening disease worldwide. Among the serogroups, there are genotypes which are designated G and P types based on the antigenic determinants of the outer layer. Distribution of the genotypes varies from place to place and more than one genotype may exist in a given geographical region. OBJECTIVE: To correlate viral load by real-time PCR in children with mild, moderate and severe rotavirus gastroenteritis assessed by the 20 point Vesikari scoring system for rotavirus diarrhea. METHODS: One hundred and twenty two faeces samples from children below 5 years of age presenting with diarrhea and positive for rotavirus by enzyme immune assay were included in the study. A 20 point Vesikari scoring system was used to objectively arrive at scoring the severity of diarrhea based on the information provided by the mothers. Twenty ‘mild’, sixty two ‘moderate’ and forty ‘severe’ category faeces samples were obtained. Samples were collected from the main hospital and from a community study. RNA extraction was done using the Qiagen kit extraction method for RNA which is a combination of silica gel based membrane and microspin technique. Reverse transcription was carried out in the presence of random primers and reverse transcriptase derived from Moloney murine leukemia virus. Real time PCR was performed on all the samples using Taqman chemistry and the crossing points (real time PCR cycle number where fluorescence crossed the threshold) obtained, which are a direct correlate of the viral load were used to study the association with severity of diarrhea. Kruskal Wallis test was the statistical method employed to study the association. RESULTS: One hundred and twenty two samples were analysed. The mean age in the community study children was less than that of the children from the hospital study. Majority of the children affected were more than 6 months of age. The median Vesikari scores in the mild, moderate and severe categories were 5, 8 and 11 respectively. There was no association observed between the viral load and severity of gastroenteritis. When associations were studied between viral load and the individual parameters of the Vesikari scoring system, there was a significant association between the number of episodes of diarrhea and the viral load. The more the number of episodes of diarrhea, the greater is the viral load. Other parameters did not have any association with viral load. CONCLUSION: There was no significant association studied between the severity of rotavirus gastroenteritis and viral load. However, with increase in the number of episodes of diarrhea, the viral load increased

    Genome sequencing for viral pathogen detection and surveillance

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