scholarly journals Targeted and Highly Multiplexed Detection of Microorganisms by Employing an Ensemble of Molecular Probes

2014 ◽  
Vol 80 (14) ◽  
pp. 4153-4161 ◽  
Author(s):  
Weihong Xu ◽  
Sujatha Krishnakumar ◽  
Molly Miranda ◽  
Michael A. Jensen ◽  
Marilyn Fukushima ◽  
...  
Keyword(s):  
Abundant Species ◽  
Laboratory Culture ◽  
Molecular Probes ◽  
Molecular Probe ◽  
Clinical Samples ◽  
Culture Independent ◽  
Order Of Magnitude ◽  
Life On Earth ◽  
Probe Set ◽  
Selection Of

ABSTRACTThe vast majority of microscopic life on earth consists of microbes that do not grow in laboratory culture. To profile the microbial diversity in environmental and clinical samples, we have devised and employed molecular probe technology, which detects and identifies bacteria that do and do not grow in culture. The only requirement is a short sequence of contiguous bases (currently 60 bases) unique to the genome of the organism of interest. The procedure is relatively fast, inexpensive, customizable, robust, and culture independent and uses commercially available reagents and instruments. In this communication, we report improving the specificity of the molecular probes substantially and increasing the complexity of the molecular probe set by over an order of magnitude (>1,200 probes) and introduce a new final readout method based upon Illumina sequencing. In addition, we employed molecular probes to identify the bacteria from vaginal swabs and demonstrate how a deliberate selection of molecular probes can identify less abundant bacteria even in the presence of much more abundant species.

scholarly journals Comparison of Common Enrichment Broths Used in Diagnostic Laboratories for Shiga Toxin—Producing Escherichia coli

2021 ◽  
Vol 9 (3) ◽  
pp. 503
Author(s):  
Michael Bording-Jorgensen ◽  
Hannah Tyrrell ◽  
Colin Lloyd ◽  
Linda Chui
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Acute Gastroenteritis ◽  
Diagnostic Testing ◽  
First Line ◽  
Gram Negative ◽  
Culture Independent ◽  
Surveillance Programs ◽  
Selection Of

Acute gastroenteritis caused by Shiga toxin-producing Escherichia coli (STEC) affects more than 4 million individuals in Canada. Diagnostic laboratories are shifting towards culture-independent diagnostic testing; however, recovery of STEC remains an important aspect of surveillance programs. The objective of this study was to compare common broth media used for the enrichment of STEC. Clinical isolates including O157:H7 as well as non-O157 serotypes were cultured in tryptic soy (TSB), MacConkey (Mac), and Gram-negative (GN) broths and growth was compared using culture on sheep’s blood agar and real-time PCR (qPCR). In addition, a selection of the same isolates was spiked into negative stool and enriched in the same three broths, which were then evaluated using culture on CHROMagarTM STEC agar and qPCR. TSB was found to provide the optimal enrichment for growth of isolates with and without stool. The results from this study suggest that diagnostic laboratories may benefit from enriching STEC samples in TSB as a first line enrichment instead of GN or Mac.

scholarly journals Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals

2020 ◽  
Author(s):  
Harsha Doddapaneni ◽  
Sara Javornik Cregeen ◽  
Richard Sucgang ◽  
Qingchang Meng ◽  
Xiang Qin ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Oligonucleotide Probe ◽  
Threshold Value ◽  
Open Reading Frames ◽  
Clinical Samples ◽  
Viral Loads ◽  
Full Length Genome ◽  
Probe Set ◽  
Reading Frames ◽  
Capture Sequencing

AbstractThe newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

scholarly journals Pseudomonas aeruginosa and Staphylococcus aureus virulence factors as biomarkers of infection

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Artur J. Sabat ◽  
Daniele Pantano ◽  
Viktoria Akkerboom ◽  
Erik Bathoorn ◽  
Alexander W. Friedrich
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Clinical Status ◽  
Clinical Samples ◽  
Pathogenic Microorganisms ◽  
Bacterial Identification ◽  
Accurate Identification ◽  
Culture Independent ◽  
And Staphylococcus Aureus

Abstract The gold standard for the diagnosis of bacterial infections in clinical samples is based on culture tests that are time-consuming and labor-intense. For these reasons, an extraordinary effort has been made to identify biomarkers as the tools for sensitive, rapid and accurate identification of pathogenic microorganisms. Moreover, biomarkers have been tested to distinguish colonization from infection, monitor disease progression, determine the clinical status of patients or predict clinical outcomes. This mini-review describes Pseudomonas aeruginosa and Staphylococcus aureus biomarkers, which contribute to pathogenesis and have been used in culture-independent bacterial identification directly from patient samples.

scholarly journals Food selection of maize weevil Sitophilus Zeamais (motschulsky)

2020 ◽  
Vol 42 (2) ◽  
Author(s):  
Nguyen Van Dzuong ◽  
Khuat Dang Long
Keyword(s):  
Food Selection ◽  
Abundant Species ◽  
Sitophilus Zeamais ◽  
Rice Grain ◽  
Maize Weevil ◽  
Food Grains ◽  
Dried Fruit ◽  
Maize Grain ◽  
Food Grain ◽  
Selection Of

The instintive behaviour exhibited by insects in the selecting  food is always a matter of  interests to entomologists, and it is one of the fundamental principles underlining the application of entomology to agriculture, horticulture and forestry. Food seclection is an important characteristic of insects that help them survive in periods with insufficient foods.Three grain types of food, maize grain, long-grain rice and soybean grain, were used in this sudy for detecting food selection behaviours of maize weevil, Sitophilus zeamais. Grains were kept in box traps put in the different stores in Son La during 90 days. The result showed that maize weevil, Sitophilus zeamais, prefered traps with maize grains (71.4%) considerably more than ones with long-grain rice (14.3%) and soybean grain (14.3%). Meanwhile, the cornsap beetle, Carpophilus dimidiatus, was mostly observed in maize grain (98.5%) and to a lesser degree, in soybean grain (1.5%); and the dried-fruit beetle, Carpophilus hemipterus, was abundantly observed in maize grain (93.1%) and less in soybean grain (6.9%). The red flour, Tribolium castaneum, also tended to select all the three food grain, i.e. maize grain: 53.5%, long-rice grain: 35.2%, and soybean grain: 11.3%.Comparing f beetle individuals captured on three types of food grains showed that the maize weevil, Sitophilus zeamais, is the most abundant species. The percentage this store beetle among all beetles observed in maize, soybean grains and long-rice grains were 57.0%, 84.9% and 27.6%, respectively. Interestingly, Ahasverus advena, foreign grain beetle, occurred abundantly on long-rice gain (61.7%), i.e. this grain is a suitable food for the development of this beetle. 

scholarly journals Culture-Independent Detection of Nontuberculous Mycobacteria in Clinical Respiratory Samples

2016 ◽  
Vol 54 (9) ◽  
pp. 2395-2398 ◽  
Author(s):  
Gianny P. Scoleri ◽  
Jocelyn M. Choo ◽  
Lex E. X. Leong ◽  
Thomas R. Goddard ◽  
Lisa Shephard ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Nontuberculous Mycobacteria ◽  
Direct Detection ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Culture Independent ◽  
Quantitative Pcr Assay ◽  
Single Reaction

Culture-based detection of nontuberculousMycobacteria(NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust.

Diagnosis of melioidosis: the role of molecular techniques

2021 ◽  
Vol 16 (4) ◽  
pp. 271-288
Author(s):  
Ian Gassiep ◽  
Delaney Burnard ◽  
Michelle J Bauer ◽  
Robert E Norton ◽  
Patrick N Harris
Keyword(s):  
Laboratory Diagnosis ◽  
Diagnostic Techniques ◽  
Molecular Techniques ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Molecular Diagnostic Techniques ◽  
Detection And Identification ◽  
Life Years ◽  
Culture Independent ◽  
Future Utility

Melioidosis is an emerging infectious disease with an estimated global burden of 4.64 million disability-adjusted life years per year. A major determinant related to poor disease outcomes is delay to diagnosis due to the fact that identification of the causative agent Burkholderia pseudomallei may be challenging. Over the last 25 years, advances in molecular diagnostic techniques have resulted in the potential for rapid and accurate organism detection and identification direct from clinical samples. While these methods are not yet routine in clinical practice, laboratory diagnosis of infectious diseases is transitioning to culture-independent techniques. This review article aims to evaluate molecular methods for melioidosis diagnosis direct from clinical samples and discuss current and future utility and limitations.

scholarly journals 655. Detection of Antibiotic Resistance Genes in Clinical Samples using T2 Magnetic Resonance

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S301-S301
Author(s):  
Jessica L Snyder ◽  
Brendan Manning ◽  
Robert Shivers ◽  
Daniel Gamero ◽  
Heidi Giese ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Magnetic Resonance ◽  
Species Identification ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Resistance Testing ◽  
Clinical Samples ◽  
Antibiotic Resistant ◽  
Blood Samples ◽  
Culture Independent

Abstract Background Antibiotic-resistant bacteria are spread through selective pressure from the use of broad-spectrum empirical therapies, mobile genetic elements that pass resistance genes between species, and the inability to rapidly and appropriately respond to their presence. Resistance gene identification is often performed with post culture molecular diagnostic tests. The T2Resistance Panel, which detects methicillin resistance genes mecA/C; vancomycin resistance genes vanA/B; carbapenemases blaKPC, blaOXA-48,blaNDM, blaVIM, and blaIMP; AmpC β-lactamases blaCMY and blaDHA; and extended-spectrum β-lactamases blaCTX-M directly from patient blood samples, is based on T2 magnetic resonance (T2MR), an FDA-cleared technology with demonstrated high sensitivity and specificity for culture-independent bacterial and fungal species identification. Here we report the clinical performance of T2MR detection of resistance genes directly from patient blood samples. Methods Patients with a clinical diagnosis of sepsis and an order for blood culture (BC) were enrolled in the study at two sites. BCs were managed using standard procedures and MALDI-TOF for species identification. Resistance testing with the T2MR assay was performed on a direct patient draw and compared with diagnostic test results from concurrent BC specimen and BC specimen taken at other points in time. The potential impact on therapy was evaluated through patient chart review. Results T2MR detected the same resistance genes as detected by post culture diagnostics in 100% of samples from concurrent blood draws. Discordant results occurred when T2MR was taken ≥48 hours after BC for patients on antimicrobial therapy. The average time to positive result was 5.9 hours with T2MR vs. 30.6 hours with post-culture molecular testing. Conclusion The T2Resistance Panel detected antibiotic resistance genes in clinical samples and displayed agreement with post culture genetic testing. T2MR results were achieved faster than culture-dependent diagnostic testing results and may allow for an earlier change from empiric to directed therapy. The use of culture-independent diagnostics like T2MR could enable a quicker response to antibiotic-resistant organisms for individual patients and developing outbreaks. Disclosures All authors: No reported disclosures.

scholarly journals Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Shunli Yang ◽  
Li Yuan ◽  
Youjun Shang ◽  
Jinyan Wu ◽  
Xiangtao Liu ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Magnetic Beads ◽  
Molecular Probes ◽  
Single Domain ◽  
Molecular Probe ◽  
Economic Losses ◽  
Single Domain Antibody ◽  
Swine Industry ◽  
E2 Protein

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10−7 and 1.35 × 10−8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

scholarly journals Ecophysiological Distinctions of Haloarchaea from a Hypersaline Antarctic Lake as Determined by Metaproteomics

2016 ◽  
Vol 82 (11) ◽  
pp. 3165-3173 ◽  
Author(s):  
Bernhard Tschitschko ◽  
Timothy J. Williams ◽  
Michelle A. Allen ◽  
Ling Zhong ◽  
Mark J. Raftery ◽  
...  
Keyword(s):  
Low Complexity ◽  
Abundant Species ◽  
Specific Protein ◽  
Organic Substrates ◽  
Deep Lake ◽  
Nitrogen And Phosphorus ◽  
Content Type ◽  
Cold Environments ◽  
Vestfold Hills ◽  
Life On Earth

ABSTRACTDeep Lake in the Vestfold Hills is hypersaline and the coldest system in Antarctica known to support microbial growth (temperatures as low as −20°C). It represents a strong experimental model because the lake supports a low-complexity community of haloarchaea, with the three most abundant species totaling ∼72%. Moreover, the dominant haloarchaea are cultivatable, and their genomes are sequenced. Here we use metaproteomics linked to metagenome data and the genome sequences of the isolates to characterize the main pathways, trophic strategies, and interactions associated with resource utilization. The dominance of the most abundant member,Halohasta litchfieldiae, appears to be predicated on competitive utilization of substrates (e.g., starch, glycerol, and dihydroxyacetone) produced byDunaliella, the lake's primary producer, while also possessing diverse mechanisms for acquiring nitrogen and phosphorus. The second most abundant member, strain DL31, is proficient in degrading complex proteinaceous matter.Hht. litchfieldiaeand DL31 are inferred to release labile substrates that are utilized byHalorubrum lacusprofundi, the third most abundant haloarchaeon in Deep Lake. The study also linked genome variation to specific protein variants or distinct genetic capacities, thereby identifying strain-level variation indicative of specialization. Overall, metaproteomics revealed that rather than functional differences occurring at different lake depths or through size partitioning, the main lake genera possess major trophic distinctions, and phylotypes (e.g., strains ofHht. litchfieldiae) exhibit a more subtle level of specialization. This study highlights the extent to which the lake supports a relatively uniform distribution of taxa that collectively possess the genetic capacity to effectively exploit available nutrients throughout the lake.IMPORTANCELife on Earth has evolved to colonize a broad range of temperatures, but most of the biosphere (∼85%) exists at low temperatures (≤5°C). By performing unique roles in biogeochemical cycles, environmental microorganisms perform functions that are critical for the rest of life on Earth to survive. Cold environments therefore make a particularly important contribution to maintaining healthy, stable ecosystems. Here we describe the main physiological traits of the dominant microorganisms that inhabit Deep Lake in Antarctica, the coldest aquatic environment known to support life. The hypersaline system enables the growth of halophilic members of theArchaea: haloarchaea. By analyzing proteins of samples collected from the water column, we determined the functions that the haloarchaea were likely to perform. This study showed that the dominant haloarchaea possessed distinct lifestyles yet formed a uniform community throughout the lake that was collectively adept at using available light energy and diverse organic substrates for growth.

scholarly journals IFCC Working Group Recommendations for Assessing Commutability Part 1: General Experimental Design

2018 ◽  
Vol 64 (3) ◽  
pp. 447-454 ◽  
Author(s):  
W Greg Miller ◽  
Heinz Schimmel ◽  
Robert Rej ◽  
Neil Greenberg ◽  
Ferruccio Ceriotti ◽  
...  
Keyword(s):  
Experimental Design ◽  
External Quality Assessment ◽  
Clinical Samples ◽  
Essential Property ◽  
Test Results ◽  
Medical Decisions ◽  
Laboratory Test Results ◽  
Fit For Purpose ◽  
Critical Components ◽  
Selection Of

Abstract Commutability is a property of a reference material (RM) that relates to the closeness of agreement between results for an RM and results for clinical samples (CSs) when measured by ≥2 measurement procedures (MPs). Commutability of RMs used in a calibration traceability scheme is an essential property for them to be fit for purpose. Similarly, commutability of trueness controls or external quality assessment samples is essential when those materials are used to assess trueness of results for CSs. This report is part 1 of a 3-part series describing how to assess commutability of RMs. Part 1 defines commutability and addresses critical components of the experimental design for commutability assessment, including selection of individual CSs, use of pooled CSs, qualification of MPs for inclusion, establishing criteria for the determination that an RM is commutable, generalization of commutability conclusions to future measurements made with the MPs included in the assessment, and information regarding commutability to be included in the certificate for an RM. Parts 2 and 3 in the series present 2 different statistical approaches to commutability assessment that use fixed criteria related to the medical decisions that will be made using the laboratory test results.

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