scholarly journals A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

2003 ◽  
Vol 17 (2) ◽  
pp. 142-146 ◽  
Author(s):  
José Freitas Siqueira Júnior ◽  
Isabela das Neves Rôças
Keyword(s):  
16S Rdna ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Nested Polymerase Chain Reaction ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root ◽  
Pcr Products ◽  
Species Specific

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

scholarly journals Performance of recombinant proteins in diagnosis and differentiation of canine visceral leishmaniasis infected and vaccinated dogs

2020 ◽  
Vol 10 (3) ◽  
pp. 165-171
Author(s):  
Ingrid E. Pereira ◽  
Kyssia P. Silva ◽  
Laura M. Menegati ◽  
Aimara C. Pinheiro ◽  
Elaine A. O. Assunção ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Recombinant Proteins ◽  
Serological Test ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Reservoir Host ◽  
Superior Performance ◽  
Canine Visceral Leishmaniasis ◽  
Roc Curve Analysis

AbstractControl of canine visceral leishmaniasis (CVL), a major zoonotic disease in Brazil and many other tropical and subtropical countries, remains difficult as an accurate and reliable diagnosis is still missing. In endemic regions, infected dogs are the main parasitic reservoir host of human Visceral leishmaniasis (VL) infection. Vaccination of dogs against Leishmania infection constitutes an important strategy to prevent or to better control CVL, thus, a serological test that can discriminate between antibodies induced by immunization versus infection is highly desirable in order to improve and simplify diagnosis. Here, four recombinant proteins were evaluated for their ability to detect and differentiate between dogs that are infected with Leishmania or have been immunized with the anti-Leishmania vaccine Leish-Tec®. Receiver operating characteristic (ROC) curve analysis of the four Leishmania-specific IgG ELISA revealed superior performance of rK28, followed by rKLO8, rK39 and rLb6H. The rK28-based ELISA revealed not only the best accuracy against CVL, but also the lowest cross-reactivity with sera from Leish-Tec® immunized dogs. Our data show that the rK28-based ELISA is highly suitable for CVL screening as it shows high sensitivity with simultaneous low cross-reactivity. Further, the high specificity of the rKLO8 indicates its suitability for the confirmation of CVL diagnosis.

scholarly journals DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

2015 ◽  
Vol 8 ◽  
pp. MBI.S29736 ◽  
Author(s):  
Kenjiro Nagamine ◽  
Guo-Chiuan Hung ◽  
Bingjie Li ◽  
Shyh-Ching Lo
Keyword(s):  
Safety Concern ◽  
Rapid Development ◽  
High Sensitivity ◽  
High Specificity ◽  
Clostridium Tetani ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Pcr Assays ◽  
Primer Sets ◽  
Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

scholarly journals Snipe: highly sensitive pathogen detection from metagenomic sequencing data

2021 ◽  
Author(s):  
Lihong Huang ◽  
Bin Hong ◽  
Wenxian Yang ◽  
Liansheng Wang ◽  
Rongshan Yu
Keyword(s):  
Foodborne Pathogens ◽  
Pathogen Detection ◽  
High Sensitivity ◽  
High Specificity ◽  
Correction Method ◽  
Food Samples ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Metagenomics Data ◽  
Species Specific

Abstract Metagenomics data provide rich information for the detection of foodborne pathogens from food and environmental samples that are mixed with complex background bacteria strains. While pathogen detection from metagenomic sequencing data has become an activity of increasing interest, shotgun sequencing of uncultured food samples typically produces data that contain reads from many different organisms, making accurate strain typing a challenging task. Particularly, as many pathogens may contain a common set of genes that are highly similar to those from normal bacteria in food samples, traditional strain-level abundance profiling approaches do not perform well at detecting pathogens of very low abundance levels. To overcome this limitation, we propose an abundance correction method based on species-specific genomic regions to achieve high sensitivity and high specificity in target pathogen detection at low abundance.

scholarly journals Comparison of Two Multiplex PCR Systems for Meat Species Authentication

2019 ◽  
Author(s):  
D. Al-taghlubee ◽  
A. Misaghi ◽  
P. Shayan ◽  
A. Akhondzadeh Basti ◽  
H. Gandomi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Cross Reactivity ◽  
Specific Pcr ◽  
Pcr Products ◽  
Reverse Primer ◽  
Specific Reverse Primer ◽  
Meat Species ◽  
Polymerase Chain ◽  
Species Authentication ◽  
Species Specific

Background: Meat species adulteration has become a problem of concern. This study aimed to compare two previously published multiplex Polymerase Chain Reaction (PCR) methods for meat species authentication.  Methods: The primers used in the first multiplex PCR involved species-specific reverse primer for sheep, goat, cattle, pig, and donkey with universal forward primer. In the second multiplex PCR, the primers included species-specific forward and reverse primer for pork, lamb, ostrich, horse, and cow. The extracted DNA was then amplified with species-specific primers and with mix primers separately in the respective multiplex PCR. Results: The first multiplex PCR was accompanied with cross reactivity, whereas the second multiplex PCR was specific as expected for pork, lamb, ostrich, horse, and cow. The first set of multiplex PCR showed not always amplification of all species-specific DNAs with a mixture of DNA from mentioned animals. Regarding the second set of primers, the extracted DNA of different meat species was amplified with corresponding species primers as simplex PCR resulting in specific amplicons for species DNA prepared from sheep, ostrich, horse, pig, and cattle with the specific PCR products of 119, 155, 253, 100, and 311 bp, respectively. Conclusion: Based on the present investigation, we recommend the multiplex PCR with the second set of primers included species-specific forward and reverse primers for species authentication of five meat types, including pork, lamb, ostrich, horse, as well as cow.

scholarly journals Snipe: Highly sensitive pathogen detection from metagenomic sequencing data

2020 ◽  
Author(s):  
Lihong Huang ◽  
Bin Hong ◽  
Wenxian Yang ◽  
Liansheng Wang ◽  
Rongshan Yu
Keyword(s):  
Foodborne Pathogens ◽  
Pathogen Detection ◽  
High Sensitivity ◽  
High Specificity ◽  
Correction Method ◽  
Food Samples ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Metagenomics Data ◽  
Species Specific

Metagenomics data provides rich information for the detection of foodborne pathogens from food and environmental samples that are mixed with complex background bacteria strains. While pathogen detection from metagenomic sequencing data has become an activity of increasing interest, shotgun sequencing of uncultured food samples typically produces data that contains reads from many different organisms, making accurate strain typing a challenging task. Particularly, as many pathogens may contain a common set of genes that are highly similar to those from normal bacteria in food samples, traditional strain-level abundance profiling approaches do not perform well at detecting pathogens of very low abundance levels. To overcome this limitation, we propose an abundance correction method based on species-specific genomic regions to achieve high sensitivity and high specificity in target pathogen detection at low abundance.

scholarly journals Performance Characteristics of Four High-Throughput Immunoassays for Detection of IgG Antibodies against SARS-CoV-2

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Elitza S. Theel ◽  
Julie Harring ◽  
Heather Hilgart ◽  
Dane Granger
Keyword(s):  
High Throughput ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Clinical Diagnostics ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Predictive Values ◽  
Serologic Tests ◽  
Convalescent Phase

ABSTRACT The role of serologic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in both the clinical and public health settings, will continue to evolve as we gain increasing insight into our immune response to the virus. Here, we evaluated four high-throughput serologic tests for detection of anti-SARS-CoV-2 IgG antibodies, from Abbott Laboratories (Abbott Park, IL), Epitope Diagnostics, Inc. (San Diego, CA), Euroimmun (Lubeck, Germany), and Ortho-Clinical Diagnostics (Rochester, NY), using a panel of serially collected serum samples (n = 224) from 56 patients with confirmed coronavirus disease 2019 (COVID-19), healthy donor sera from 2018, and a cross-reactivity serum panel collected in early 2020. The sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays in convalescent-phase serum samples collected more than 14 days post-symptom onset or post-initial positive reverse transcriptase PCR (RT-PCR) result were 92.9% (78/84), 88.1% (74/84), 97.6% (82/84), and 98.8% (83/84), respectively. Among unique convalescent patients, sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays were 97.3% (36/37), 73% (27/37), 94.6% (35/37), and 97.3% (36/37), respectively. Overall assay specificity/positive predictive values based on a 5% prevalence rate were 99.6%/92.8%, 99.6%/90.6%, 98.0%/71.2%, and 99.6%/92.5%, respectively, for the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays. In conclusion, we show high sensitivity in convalescent-phase sera and high specificity for the Abbott, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays. With the unprecedented influx of commercially available serologic tests for detection of antibodies against SARS-CoV-2, it remains imperative that laboratories thoroughly evaluate such assays for accuracy prior to implementation.

scholarly journals Fluorimetric Immunoassay for Multianalysis of Aflatoxins

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Lizy Kanungo ◽  
Sunil Bhand
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Standard Addition ◽  
Cross Reactivity ◽  
Addition Method ◽  
Milk Samples ◽  
Capture Antibody ◽  
Multianalyte Detection ◽  
Microwell Plate ◽  
Detection And Quantification

A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25–50 pg/mL. AFM1 as low as 1 pg/mL was detected by this method with assay volume 40 μL. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.

scholarly journals Molecular detection and identification of Enterococcus faecium isolated from dental root canals

2021 ◽  
pp. 1447-1451
Author(s):  
Eman A. Mustafa ◽  
Suhad M. Hamdoon ◽  
Enas Y. Shehab
Keyword(s):  
Root Canal ◽  
Enterococcus Faecium ◽  
Brain Heart Infusion ◽  
Traditional Culture ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Culture Methods ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root

Enterococci are usually encountered and predominate in oral infections, especially those associated with dental root canal infections of necrotic pulp and periodontitis. This study aimed to detect and identify Enterococcus faecium isolated from infected root canals, using polymerase chain reaction ( PCR). Thirty samples were collected from patients with  necrotic pulp, infected root canals, and endodontic treatment failure, attending the Conservative Treatment Department, College of Dentistry, Mosul University, Dental Teaching Hospital. The samples were obtained by inserting sterile paper points into the root canals and transferred in brain heart infusion broth vials to be  inoculated in a selective M-Enterococcus Agar Base . Twenty five isolates that belong to the genus Enterococcus were   recognized by traditional culture methods and biochemical tests. Then, DNA extractions of these isolates were carried out for  identification  with PCR by the amplification of ddI (D-Ala-D-Ala Ligase) chromosomal genes of Enterococcus faecium. Among the 25  isolates, twenty (80%) were identified to the level of Enterococcus faecium by traditional culture methods and biochemical tests, in comparison to 17 (68%) identified by  molecular identification. The PCR products for the specific primer produced bands on agarose gel at the position of 658bp. The study showed that the use of PCR with primers for the E. faecium ddI gene may be the most accurate method for rapid identification of Enterococci. Molecular identification of Enterococcus spp. revealed a significant role of E. feacium in root canal infections. Also, the detection of ddI gene  using PCR provides a definitive target that could be used for the detection of E. faecium  from clinical samples.

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