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PubMedDate not supplied

Feature extraction from light-scatter patterns of Listeria colonies for identification and classification.

Bacterial contamination by Listeria monocytogenes not only puts the public at risk, but also is costly for the food-processing industry. Traditional biochemical methods for pathogen identification require complicated sample preparation for reliable results. Optical scattering technology has been used for identification of bacterial cells in suspension, but with only limited success. Therefore, to improve the efficacy of the identification process using our novel imaging approach, we analyze bacterial colonies grown on solid surfaces. The work presented here demonstrates an application of computer-vision and pattern-recognition techniques to classify scatter patterns formed by Listeria colonies. Bacterial colonies are analyzed with a laser scatterometer. Features of circular scatter patterns formed by bacterial colonies illuminated by laser light are characterized using Zernike moment invariants. Principal component analysis and hierarchical clustering are performed on the results of feature extraction. Classification using linear discriminant analysis, partial least squares, and neural networks is capable of separating different strains of Listeria with a low error rate. The demonstrated system is also able to determine automatically the pathogenicity of bacteria on the basis of colony scatter patterns. We conclude that the obtained results are encouraging, and strongly suggest the feasibility of image-based biodetection systems.

Algorithms↗
PubMed2005

Development of a DNA microarray for detection and identification of fungal pathogens involved in invasive mycoses.

Invasive fungal infections have emerged as a major cause of morbidity and mortality in immunocompromised patients. Conventional identification of pathogenic fungi in clinical microbiology laboratories is time-consuming and, therefore, often imperfect for the early initiation of an adequate antifungal therapy. We developed a diagnostic microarray for the rapid and simultaneous identification of the 12 most common pathogenic Candida and Aspergillus species. Oligonucleotide probes were designed by exploiting the sequence variations of the internal transcribed spacer (ITS) regions of the rRNA gene cassette to identify Candida albicans, Candida dubliniensis, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. By using universal fungal primers (ITS 1 and ITS 4) directed toward conserved regions of the 18S and 28S rRNA genes, respectively, the fungal ITS target regions could be simultaneously amplified and fluorescently labeled. To establish the system, 12 pre-characterized fungal strains were analyzed; and the method was validated by using 21 clinical isolates as blinded samples. As the microarray was able to detect and clearly identify the fungal pathogens within 4 h after DNA extraction, this system offers an interesting potential for clinical microbiology laboratories.

Aspergillosis↗
PubMed1998

[Diagnostic molecular microbiology--identification of Staphylococcus epidermidis].

The species Staphylococcus epidermidis is the predominant coagulase-negative staphylococci (CNS) isolated from clinical sources. S. epidermidis is now recognized as an important nosocomial pathogen. Identification of CNS is often performed using diagnostic kits based on biochemical or immunological reactions. However, these kits are often unreliable for the identification of CNS species including S. epidermidis. Currently, ribosomal RNA (rRNA) analyses are the most powerful methods for determining phylogenetic relationships among microorganisms and also for identification of species. Several aspects of construction of ribosomal probes for identification of CNS species are presented and discussed. Additionally, the application of restriction fragment length polymorphisms (RFLP) of rRNA genes for differentiation of clinical isolates of S. epidermidis is shown.

Cross Infection↗
PubMed2026

Molecular Diagnostics for WHO Priority Bacterial Pathogens: A Bibliometric Mapping of Diagnostic Platforms, Resistance Markers, and Antimicrobial Resistance Research Trends.

Antimicrobial resistance (AMR) constrains effective treatment and carries implications for infection control, surveillance, and public health. The World Health Organization (WHO) priority bacterial pathogen framework has intensified the need for diagnostic innovation by redefining research priorities around organisms combining high disease burden with complex resistance profiles. Molecular diagnostics have accordingly moved beyond culture-based workflows, integrating rapid pathogen identification, resistance-marker detection, genomic surveillance, and clinical decision support. The present study conducted a bibliometric mapping of the literature on WHO priority pathogens. Rather than addressing resistance at a general level or a single pathogen or technology, it integrates priority pathogens, molecular platforms, and resistance markers within a single framework, tracing their joint thematic and temporal evolution along an explicit pathogen-platform-marker axis. Scopus-indexed articles and reviews (2000-2025) were retrieved, yielding 1746 publications after screening adapted from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Analyses used Bibliometrix/Biblioshiny, R, and VOSviewer. The literature expanded markedly after 2018, led by China and the United States. Methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis, Enterococcus faecium, and the Enterobacterales-carbapenemase axis constituted the principal thematic cores, whereas conventional polymerase chain reaction (PCR)/nucleic acid amplification testing (NAAT) and whole-genome sequencing were the dominant platforms. Overall, the field has evolved from pathogen detection into an AMR-centered translational domain encompassing resistance prediction, genomic epidemiology, surveillance, and clinical decision support. Diagnostic development, stewardship, and surveillance depend on hybrid workflows coupling rapid marker-targeted assays with genome-based characterization, delivering actionable resistance within clinically meaningful timeframes, and extending coverage to underrepresented pathogens and platforms.

Humans↗
PubMed2026

The diagnostic potential of combined quantitative polymerase chain reaction and next-generation sequencing using the same primers for periprosthetic joint infection.

Next-generation sequencing (NGS) enables the detection of specific pathogens unidentifiable by conventional cultures, but its application in orthopedics remains inconsistent due to background contamination and irreproducible findings. This study evaluated the diagnostic performance of a novel workflow combining broad-range 16S rRNA gene quantitative PCR (qPCR) screening with downstream NGS, focusing on bacterial biomass thresholds. The qPCR assay demonstrated excellent intrarater reliability, with an intraclass correlation coefficient (ICC) of 0.961 (95% confidence interval, 0.881 to 0.997). Based on serially diluted positive controls, a quantitative threshold of 10⁵ CFU/mL was established as the minimum concentration required for the consistent detection of fastidious taxa, such as Escherichia coli. When evaluated against conventional cultures using 95 sonicate fluid and 276 pre/intraoperative tissue samples, the qPCR assay achieved a sensitivity of 80% and a specificity of 72%. Subsequent NGS sequencing of 26 clinical samples and 9 controls showed concordance in 4 of 6 culture-positive infected cases with NGS taxonomy, whereas the remaining discrepancies were likely attributable to culture-based phenotypic misidentification. Notably, among the qPCR-positive cases, three were culture-negative, including two hip prosthesis loosening cases exhibiting polymicrobial profiles, and one post-traumatic osteoarthritis case harboring low-level Staphylococcus. Crucially, this post-traumatic patient developed delayed periprosthetic joint infection (PJI) 2 years post-surgery, with cultures identifying Staphylococcus previously detected by the initial NGS analysis. Integrating qPCR screening with targeted NGS effectively refines pathogen identification, filters environmental artifacts, and overcomes the diagnostic limitations of culture-negative infections in orthopedic practice.IMPORTANCENext-generation sequencing (NGS) enables the detection of specific pathogens in clinical samples that are not identifiable by conventional methods. However, NGS applications in orthopedics have not been quantitatively evaluated, and findings have been inconsistent owing to contaminants and the presence of non-credible causative organisms. These factors primarily stem from the failure to evaluate low-biomass samples and the absence of proper controls, such as negative controls or mock community DNA samples. This study demonstrates that interpreting results from low-biomass samples requires careful consideration because NGS relies on relative bacterial abundances; distinguishing likely pathogens from contaminants is particularly challenging when bacterial loads are low. We demonstrated that combining NGS with quantitative PCR (qPCR) and applying a Cq cutoff can reduce false positives.

Humans↗
PubMed2026

TaxTriage: an open-source metagenomic sequencing data analysis pipeline enabling putative pathogen detection.

MOTIVATION: TaxTriage is a comprehensive pathogen identification workflow designed for both short- and long-read untargeted DNA and RNA sequencing data. Combining read classification, mapping, and de novo assembly approaches, putative pathogens are identified through comparisons to curated pathogens and abundance expectations from healthy cohort data. Flexible installation options are enabled using Nextflow™ (NF), including cloud deployment via NF Tower (Seqera Platform) and local installation on a variety of systems, including standalone installations without external internet access. Final analysis summaries are compiled into an Organism Discovery Report, which lists likely pathogens and supporting data, including a custom confidence score. RESULTS: Evaluation of published in silico, clinical, and outbreak datasets identified performance comparable to alternative cloud-based processing pipelines for expected pathogen and co-infection detection with similar sensitivity and increased specificity. To support both public health and veterinary diagnostics communities, customization options have been incorporated to enable improved performance for host species of interest. AVAILABILITY AND IMPLEMENTATION: Source code for TaxTriage is freely available at https://github.com/jhuapl-bio/taxtriage. TaxTriage v2.1.1 has been archived on Zenodo at https://zenodo.org/records/17081354 to permit reproducible analysis as described in this manuscript.

Software↗
PubMed2006

Rapid identification, virulence analysis and resistance profiling of Staphylococcus aureus by gene segment-based DNA microarrays: application to blood culture post-processing.

Up to now, blood culturing systems are the method of choice to diagnose bacteremia. However, definitive pathogen identification from positive blood cultures is a time-consuming procedure, requiring subculture and biochemical analysis. We developed a microarray for the identification of Staphylococcus aureus comprising PCR generated gene-segments, which can reduce the blood culture post-processing time to a single day. Moreover, it allows concomitant identification of virulence factors and antibiotic resistance determinants directly from positive blood cultures without previous amplification by PCR. The assay unambiguously identifies most of the important virulence genes such as tsst-1, sea, seb, eta and antibiotic resistance genes such as mecA, aacA-aphD, blaZ and ermA. To obtain positive signals, 20 ng of purified genomic S. aureus DNA or 2 microg of total DNA extracted from blood culture was required. The microarray specifically distinguished S. aureus from gram-negative bacteria as well as from closely related coagulase negative staphylococci (CoNS). The microarray-based identification of S. aureus can be accomplished on the same day blood cultures become positive in the Bactec. The results of our study demonstrate the feasibility of microarray-based systems for the direct identification and characterization of bacteria from cultured clinical specimens.

Bacterial Proteins↗
PubMed2003

Determination of the frequency of inadequate antibiotic therapy using calculation of indication failure (IF), cumulative indication failure (CIF), and balanced indication failure (BIF).

Because results of pathogen identification are often lacking when antibiotic therapy is initiated, treatment must frequently be instituted on an empirical basis. The type of empirical therapy will depend on the anticipated pathogen spectrum and naturally also on the prevailing resistance patterns. Inadequate antibiotic therapy may not only be associated with increased overall treatment costs, but will also have adverse effects on mortality. The clinician is frequently faced with an overabundant variety of microbiological data and may fail to interpret them correctly. Therefore, the present study has attempted to "translate" the available microbiological resistance data, frequently presented in the form of percentage rates, into concrete patient numbers and thus illustrate the frequency of inadequate antibiotic therapy. For this purpose, "Indication Failure" (IF), "Cumulative Indication Failure" (CIF) and "Balanced Indication Failure" (BIF) have been calculated based on available microbiological data. For the indication "nosocomial pneumonia", calculations of the BIF show that only one out of 67 or one out of 63 patients is inadequately treated with a therapy with cefepime or imipenem, while one out of 25 patients is inadequately treated when using ceftazidime. However, it must be pointed out that these calculations only represent an interpretation of microbiological data and the success of antibiotic therapy will ultimately also depend on parameters such as the pharmacodynamic properties of an antibiotic or on the immunocompetence of the patient treated.

Anti-Bacterial Agents↗
PubMed2002

Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences.

The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated. A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae. Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation. This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods. Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity). Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems.

Base Sequence↗
PubMed2026

Rapid diagnosis of common, undetected, and uncultivable bloodstream infections from positive blood cultures using Oxford Nanopore sequencing: a metagenomic pipeline analysis.

BACKGROUND: Metagenomic sequencing can potentially transform clinical microbiology by enabling rapid pathogen identification and antimicrobial resistance (AMR) prediction in critically ill patients with bloodstream infections. However, the clinical use of metagenomic sequencing has been constrained by its speed, accuracy, and technical feasibility. Our aim was to develop and evaluate a direct-from-positive blood culture workflow using Oxford Nanopore sequencing that overcomes these limitations and delivers rapid, accurate results. METHODS: In this metagenomic pipeline analysis, 211 positive (130 aerobic and 81 anaerobic) and 62 negative (30 aerobic and 32 anaerobic) randomly selected blood cultures were processed from Oxford University Hospitals for comparing species identification, AMR detection, and time-to-result against standard culture-based diagnostics performed by the hospital's routine microbiology laboratory. Species prediction was performed using Kraken2 with a comprehensive standard database, applying heuristic and random forest classification models. Additionally, we benchmarked AMR classification tools and databases, including ResFinder, CARD, and NCBI AMRFinderPlus. FINDINGS: Across all samples, our method achieved 97% sensitivity and 94% specificity for species identification compared with that of routine culture and matrix-assisted laser desorption ionisation time-of-flight-based diagnostics; both sensitivity and specificity increased to 100% after adjudication of plausible additional infections. We detected 19 additional infections (13 polymicrobial, five previously unidentifiable, and one in a culture-negative sample) and delivered species identification results within 3 h 20 min (IQR 3 h 7 min-3 h 27 min), approximately 10 h earlier than routine diagnostic methods. For the ten most common clinically relevant pathogens, our method yielded AMR results 20 h earlier than current antimicrobial susceptibility testing, with an overall sensitivity of 88% and specificity of 93%. Performance varied by species. For Staphylococcus aureus, the AMR prediction sensitivity was 100% and specificity was 99%, and for Escherichia coli, the prediction sensitivity was 91% and specificity was 94%. INTERPRETATION: These findings show that metagenomic sequencing has the potential to rapidly and comprehensively detect pathogens and AMR in bloodstream infections. Integration into clinical practice could help to close diagnostic gaps, reduce empirical antibiotic use, and enable rapid targeted treatment. Nonetheless, improvements in AMR prediction for some species and drugs, along with further multisite validation, are required before clinical implementation. FUNDING: National Institute for Health Research (NIHR) Oxford Biomedical Research Centre.

Humans↗
PubMed2006

Single-stage treatment of pyogenic spinal infection with titanium mesh cages.

STUDY DESIGN: Single institution retrospective review. OBJECTIVES: To report a series of pyogenic spinal infections treated with single-stage debridement and reconstruction with titanium mesh cages. SUMMARY OF BACKGROUND DATA: Various studies have reported surgical results of pyogenic spinal osteomyelitis with anterior debridement, strut grafting and fusion, including delayed posterior spinal instrumentation. Additionally, various authors have recommended against the use of instrumentation because of the concern about glycocalyx formation on the metal and chronic infection. At our institution, we routinely treat chronic vertebral osteomyelitis with single-stage debridement, reconstruction with a titanium mesh cage filled with allograft chips and demineralized bone matrix, and posterior pedicle screw instrumentation. To our knowledge, this is the largest single series reporting single-stage debridement and instrumentation of pyogenic spinal infection with titanium mesh cages and posterior instrumentation. MATERIALS AND METHODS: We retrospectively reviewed the patient records and radiographs of 21 consecutive patients (average age 49.3 years, range 23 to 80 years) with pyogenic vertebral osteomyelitis, all treated with titanium mesh cages. Average follow-up was 44 months (range, 25 to 70 months). Spinal levels included 6 thoracic, 4 thoracolumbar, 9 lumbar, and 2 lumbosacral (L5-S1) lesions. All patients had preoperative serum evaluation, which usually included blood cultures, complete blood count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), in addition to plain radiographs and magnetic resonance imaging. A positive needle biopsy was available in only 2/7 patients (29%), and overall, preoperative pathogen identification was available in only 7/21 patients (33%). All patients were treated postoperatively with a minimum of 6 weeks of intravenous antibiotics, with a specific antibiotic regimen directed toward the postoperative pathogen when identified (17/21 cases). Extensive radiographic evaluation was also performed. RESULTS: ESR and CRP were routinely elevated (18/20 and 11/17 cases respectively), whereas the white blood count was elevated in only 8 out of 21 cases (38%). The average duration of symptoms to diagnosis was approximately 13.6 weeks (range 3 weeks to 10 months). The indications for surgery included neurologic compromise, significant vertebral body destruction with loss of sagittal alignment, failure of medical treatment, and/or epidural abscess. All patients had resolution of infection, as noted by normalization of the ESR and CRP. Further, 16 out of 21 patients also had a significant reduction of pain. There were no deaths or new postoperative neurologic compromise. The most common pathogen was Staphylococcus aureus. Two patients required a second surgery (posterior irrigation and debridement) during the same admission for persistent wound drainage. Radiographically, the average segmental kyphosis (or loss of lordosis) was 11.5 degrees (range, 0 to 24 degrees) preoperatively, and +0.8 degrees (range, -3 to +5 degrees) at latest postoperative follow-up. There was an average of 2.2 mm cage settling (range, 0 to 5 mm) on latest follow-up. There were no instrumentation failures, signs of chronic infection, or rejection. CONCLUSIONS: Titanium mesh cages present a viable option for single-stage anterior surgical debridement and reconstruction of vertebral osteomyelitis, without evidence of chronic infection or rejection. When used in conjunction with pedicle screw instrumentation, there is minimal cage settling without loss of sagittal alignment.

Adult↗
PubMed2002

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene.

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.

Aged↗
PubMed2005

Rostraureum tropicale gen. sp. nov. (Diaporthales) associated with dying Terminalia ivorensis in Ecuador.

Terminalia ivorensis, a tree of central African origin, is planted in several tropical countries for timber and veneer production. During the course of a recent disease survey, an unknown fungus was found associated with basal cankers on dying T. ivorensis in Ecuador. The fungus has orange fruiting structures and septate, fusoid ascospores, similar to those of Cryphonectria, a well-known genus of canker pathogens. The aim of this study was to identify the fungus and to assess its pathogenicity. Identification was based on morphological characteristics as well as DNA sequence data. DNA sequence data from the ITS regions of the rDNA operon and two regions of the beta-tubulin gene, were compared with published sequences of Cryphonectria species and the closely related genera Endothia and Chrysoporthe. Pathogenicity tests were conducted on T. superba saplings. Morphological characterisations revealed that the conidiomata of the fungus from T. ivorensis, differed from those typical of Cryphonectria in being superficial and rostrate. Only Cryphonectria longirostris was similar to the fungus from T. ivorensis, but could be distinguished from it based on conidial size. Phylogenetic analyses showed that the fungus from T. ivorensis grouped closely with species of Cryphonectria, Chrysoporthe and Endothia, yet formed a distinct clade. Pathogenicity tests on T. superba provided evidence that the fungus is able to cause distinct stem cankers. We conclude that the pathogenic fungus from T. ivorensis represents a new genus and new species in the Diaporthales and we provide the name Rostraureum tropicale for it. The genus is typified by R. tropicale. Furthermore, C. longirostris is transferred to Rostraureum.

Ascomycota↗
PubMed2001

Urinary tract infections in children younger than 5 years of age: epidemiology, diagnosis, treatment, outcomes and prevention.

Although the true incidence of urinary tract infections (UTIs) in children is difficult to estimate, they are one of the most common bacterial infections seen by clinicians who care for young children. Except for the first 8 to 12 weeks of life, when infection of the urinary tact may be secondary to a haematogenous source, UTI is believed to arise by the ascending route after entry of bacteria via the urethra. Enterobacteriaceae are the most common organisms isolated from uncomplicated UTI. Infection with Staphylococcus aureus is rare in children without in-dwelling catheters or other sources of infection, and coagulase-negative staphylococci and Candida spp. are associated with infections after instrumentation of the urinary tract. The diagnosis of UTI in young children is important as it is a marker for urinary tract abnormalities and, in the newborn, may be associated with bacteraemia. Early diagnosis is critical to preserve renal function of the growing kidney. A urine specimen for culture is necessary to document a UTI in a young child. Prior to culture, urinalysis may be useful to detect findings supporting a presumptive diagnosis of UTI. The goals of the management of UTI in a young child are: (i) prompt diagnosis of concomitant bacteraemia or meningitis, particularly in the infant; (ii) prevention of progressive renal disease by prompt eradication of the bacterial pathogen, identification of abnormalities of the urinary tract and prevention of recurrent infections; and (iii) resolution of the acute symptoms of the infection. Delay in initiation of the antibacterial therapy is associated with an increased risk of renal scarring. The initial choice of antibacterial therapy is based on the knowledge of the predominant pathogens in the patient's age group, antibacterial sensitivity patterns in the practice area, the clinical status of the patient and the opportunity for close follow-up. Imaging studies to detect congenital or acquired abnormalities are recommended following the first UTI in all children aged <6 years. Patients with significant urinary tract abnormalities and/or frequent symptomatic UTI may benefit from prophylactic antibacterials. The main long term consequence of UTI is renal scarring which may lead to hypertension and end-stage renal disease. Prevention of recurrent UTI focuses on detection, and correction if possible, of urinary tract abnormalities. Interventions that have been associated with a decrease in symptomatic UTI in children with a history of recurrent UTI include relief of constipation and voiding dysfunction.

Anti-Infective Agents, Urinary↗
PubMed2003

PCR methodology as a valuable tool for identification of endodontic pathogens.

OBJECTIVES: This paper reviews the principles of polymerase chain reaction (PCR) methodology, its application in identification of endodontic pathogens and the perspectives regarding the knowledge to be reached with the use of this highly sensitive, specific and accurate methodology as a microbial identification test. DATA SOURCES: Studies published in the medical, dental and biological literature. STUDY SELECTION: Evaluation of published epidemiological studies examining the endodontic microbiota through PCR methodology. CONCLUSIONS: PCR technology has enabled the detection of bacterial species that are difficult or even impossible to culture as well as cultivable bacterial strains showing a phenotypically divergent or convergent behaviour. Moreover, PCR is more rapid, much more sensitive, and more accurate when compared with culture. Its use in endodontics to investigate the microbiota associated with infected root canals has expanded the knowledge on the bacteria involved in the pathogenesis of periradicular diseases. For instance, Tannerella forsythensis (formerly Bacteroides forsythus), Treponema denticola, other Treponema species, Dialister pneumosintes, and Prevotella tannerae were detected in infected root canals for the first time and in high prevalence when using PCR analysis. The diversity of endodontic microbiota has been demonstrated by studies using PCR amplification, cloning and sequencing of the PCR products. Moreover, other fastidious bacterial species, such as Porphyromonas endodontalis, Porphyromonas gingivalis and some Eubacterium spp., have been reported in endodontic infections at a higher prevalence than those reported by culture procedures.

Bacteria↗
PubMed2004

Identification of Francisella tularensis using real-time fluorescence polymerase chain reaction.

A Francisella tularensis-specific, TaqMan probe-based, real-time fluorescence polymerase chain reaction (PCR) assay required approximately 60 minutes and consistently achieved a sensitivity of < or = 10 fg of F. tularensis genomic DNA (five genome equivalents). Specificity testing against a genomic DNA cross-reaction panel comprised of 22 bacterial organisms representing closely related species, diverse genera, and human genomic DNA resulted in no false positives of significance. The assay was conducted on a field-deployable thermocycler, the R.A.P.I.D. ("Ruggedized" Advanced Pathogen Identification Device), a microbial identification system that can provide rapid and accurate identification F. tularensis.

Animals↗
PubMed2006

Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing.

Rapid detection and identification of influenza virus is becoming increasingly important in the face of concerns over an influenza pandemic. A fully integrated and self-contained microfluidic device has been developed to rapidly identify influenza A hemagglutinin and neuraminidase subtypes and sequence portions of both genes. The device consists of a DNA microarray with 12 000 features and a microfluidic cartridge that automates the fluidic handling steps required to carry out a genotyping assay for pathogen identification and sequencing. The fully integrated microfluidic device consists of microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross talk of the stored reagents. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows the detection and identification of influenza virus in a rapid and automated fashion.

DNA, Viral↗
PubMed2002

Plant disease resistance: commonality and novelty in multicellular innate immunity.

Pathogen avirulence genes encode for effector molecules that play a crucial role in the process of pathogen colonization of plant tissue. Successful host defense requires rapid and efficient detection of the pathogen avirulence factors. In the last few years, much progress has been made in delineating the plant molecular sentinels that participate in pathogen identification. Because this ability is genetic information that is 'hard-wired' into the genome, it is called 'innate immunity' and it draws its origins from a phylogenetically ancient form of immunity common to plants and animals. Conservation is shown in many of the functional molecular motifs of innate genes such as the Toll/interleukin 1 receptor domains, nucleotide binding domains and structures that contain leucine rich repeats. Novel plant molecular surveillance domains also include pathogen pattern recognition by coiled-coil domains and specialized kinases. The rapid evolution of plant innate immunity genes is readily detected in their sequence polymorphism, by their massive amplification and appearance in the genome in a clustered organization. By comparative biology of highly diverged innate immunity systems we can enhance our appreciation of the truly basic forces that have shaped its evolution in mutlicellular organisms.

Amino Acid Sequence↗