scholarly journals Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

2019 ◽  
Vol 5 (4) ◽  
pp. 98 ◽  
Author(s):  
Rita Caramalho ◽  
Lisa Madl ◽  
Katharina Rosam ◽  
Günter Rambach ◽  
Cornelia Speth ◽  
...  
Keyword(s):  
Growth Stage ◽  
Limit Of Detection ◽  
Fungal Infections ◽  
Large Subunit ◽  
Mucor Circinelloides ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Species Complexes ◽  
Pure Cultures ◽  
Sensitivity Specificity

Mucormycosis infections are infrequent yet aggressive and serious fungal infections. Early diagnosis of mucormycosis and its discrimination from other fungal infections is required for targeted treatment and more favorable patient outcomes. The majority of the molecular assays use 18 S rDNA. In the current study, we aimed to explore the potential of the mitochondrial rnl (encoding for large-subunit-ribosomal-RNA) gene as a novel molecular marker suitable for research and diagnostics. Rnl was evaluated as a marker for: (1) the Mucorales family, (2) species identification (Rhizopus arrhizus, R. microsporus, Mucor circinelloides, and Lichtheimia species complexes), (3) growth stage, and (4) quantification. Sensitivity, specificity, discriminatory power, the limit of detection (LoD), and cross-reactivity were evaluated. Assays were tested using pure cultures, spiked clinical samples, murine organs, and human paraffin-embedded-tissue (FFPE) samples. Mitochondrial markers were found to be superior to nuclear markers for degraded samples. Rnl outperformed the UMD universal® (Molyzm) marker in FFPE (71.5% positive samples versus 50%). Spiked blood samples highlighted the potential of rnl as a pan-Mucorales screening test. Fungal burden was reproducibly quantified in murine organs using standard curves. Identification of pure cultures gave a perfect (100%) correlation with the detected internal transcribed spacer (ITS) sequence. In conclusion, mitochondrial genes, such as rnl, provide an alternative to the nuclear 18 S rDNA genes and deserve further evaluation.

scholarly journals Identification of Acinetobacter baumannii and its carbapenem-resistant gene blaOXA-23-like by multiple cross displacement amplification combined with lateral flow biosensor

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shoukui Hu ◽  
Lina Niu ◽  
Fan Zhao ◽  
Linlin Yan ◽  
Jinqing Nong ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Conventional Culture ◽  
Carbapenem Resistant ◽  
Multiple Cross ◽  
Pure Cultures ◽  
Amplification Technique ◽  
Sensitivity Specificity

AbstractAcinetobacter baumannii is a frequent cause of the nosocomial infections. Herein, a novel isothermal amplification technique, multiple cross displacement amplification (MCDA) is employed for detecting all A. baumannii strains and identifying the strains harboring blaOXA-23-like gene. The duplex MCDA assay, which targets the pgaD and blaOXA-23-like genes, could identify the A. baumannii isolates and differentiate these isolates harboring blaOXA-23-like gene. The disposable lateral flow biosensors (LFB) were used for analyzing the MCDA products. A total of sixty-eight isolates, include fifty-three A. baumannii strains and fifteen non-A. baumannii strains, were employed to optimize MCDA methods and determine the sensitivity, specificity and feasibility. The optimal reaction condition is found to be 63 °C within 1 h, with limit of detection at 100 fg templates per tube for pgaD and blaOXA-23-like genes in pure cultures. The specificity of this assay is 100%. Moreover, the practical application of the duplex MCDA-LFB assay was evaluated using clinical samples, and the results obtained from duplex MCDA-LFB method were consistent with conventional culture-based technique. In sum, the duplex MCDA-LFB assay appears to be a reliable, rapid and specific technique to detect all A. baumannii strains and identify these strains harboring blaOXA-23-like gene for appropriate antibiotic therapy.

scholarly journals Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections

2020 ◽  
Vol 6 (4) ◽  
pp. 308
Author(s):  
Joana Carvalho-Pereira ◽  
Filipa Fernandes ◽  
Ricardo Araújo ◽  
Jan Springer ◽  
Juergen Loeffler ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Correct Identification ◽  
Colony Pcr ◽  
Pcr Multiplex ◽  
Pathogen Dna ◽  
Species Specific

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

scholarly journals Fluorescence Polarization Immunoassay for Determination of Enrofloxacin in Pork Liver and Chicken

Molecules ◽  
2019 ◽  
Vol 24 (24) ◽  
pp. 4462
Author(s):  
Xing Shen ◽  
Jiahong Chen ◽  
Shuwei Lv ◽  
Xiulan Sun ◽  
Boris B. Dzantiev ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
Limit Of Detection ◽  
Polyclonal Antibodies ◽  
Screening Method ◽  
Sample Pretreatment ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Fluorescence Polarization Immunoassay ◽  
Pork Liver ◽  
Sensitivity Specificity

Enrofloxacin (ENR) is a widely used fluoroquinolone (FQ) antibiotic for antibacterial treatment of edible animal. In this study, a rapid and highly specific fluorescence polarization immunoassay (FPIA) was developed for monitoring ENR residues in animal foods. First, ENR was covalently coupled to bovine serum albumin (BSA) to produce specific polyclonal antibodies (pAbs). Three fluorescein-labeled ENR tracers (A, B, and C) with different spacers were synthesized and compared to obtain higher sensitivity. Tracer C with the longest arm showed the best sensitivity among the three tracers. The developed FPIA method showed an IC50 (50% inhibitory concentration) of 21.49 ng·mL−1 with a dynamic working range (IC20–IC80) of 4.30–107.46 ng·mL−1 and a limit of detection (LOD, IC10) of 1.68 ng·mL−1. The cross-reactivity (CR) of several structurally related compounds was less than 2%. The recoveries of spiked pork liver and chicken samples varied from 91.3% to 112.9%, and the average coefficients of variation were less than 3.83% and 5.13%, respectively. The immunoassay took only 8 min excluding sample pretreatment. This indicated that the established method had high sensitivity, specificity, and the advantages of simplicity. Therefore, the proposed FPIA provided a useful screening method for the rapid detection of ENR residues in pork liver and chicken.

scholarly journals Comparison of commercial RT-PCR diagnostic kits for COVID-19

2020 ◽  
Author(s):  
Puck B. van Kasteren ◽  
Bas van der Veer ◽  
Sharon van den Brink ◽  
Lisa Wijsman ◽  
Jørgen de Jonge ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Cross Reactivity ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Specific Method ◽  
Preliminary Evaluation ◽  
Rt Pcr ◽  
Accurate Identification ◽  
Clinical Sensitivity ◽  
Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

scholarly journals Polymethacrylate Sphere-Based Assay for Ultrasensitive miRNA Detection

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Samira Hosseini ◽  
Patricia Vázquez-Villegas ◽  
Richard C. Willson ◽  
Marco Rito-Palomares ◽  
Margarita Sanchez-Dominguez ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Breast Cancer Incidence ◽  
High Specific Surface Area ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Important Target ◽  
Target Analytes ◽  
High Sequence Homology ◽  
Mirna Detection ◽  
Sensitivity Specificity

Although microRNAs (miRNAs) have emerged as increasingly important target analytes, their biorecognition remains challenging due to their small size, high sequence homology, and low abundance in clinical samples. Nanospheres and microspheres have also gained increasing attention in biosensor applications due to their high specific surface area and the wide variety of compositions available. In this study, chemically designed and synthesized microspheres with active functional groups were used to promote effective miRNA immobilization resulting in better biorecognition. Upon conjugation with fluorescence-labeled complimentary probes, acylate-based spheres have indirectly detected MiR159, offering significantly enhanced analytical sensitivity, specificity, and accuracy while yielding a considerably low limit of detection (LOD) of 40 picomolar. Furthermore, MiR159 presence, which is known to be inversely correlated to breast cancer incidence and progression, was successfully detected in a competitive assay, which is promising for upgrading the current assay to clinical use.

scholarly journals Development of a highly sensitive magneto-enzyme lateral flow immunoassay for dengue NS1 detection

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7779 ◽  
Author(s):  
Tien V. Tran ◽  
Ba V. Nguyen ◽  
Thao T.P. Nguyen ◽  
Tung T. Tran ◽  
Khanh G. Pham ◽  
...  
Keyword(s):  
Test Performance ◽  
Limit Of Detection ◽  
Dengue Infection ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
The Novel ◽  
Structural Protein ◽  
Highly Sensitive

Background Dengue infection represents a global health issue of growing importance. Dengue non-structural protein 1 (NS1) plays a central role in the early detection of the disease. The most common method for NS1 detection is testing by lateral flow immunoassays (LFIAs) with varying sensitivity. In this study, we present a highly sensitive magneto-enzyme LFIA for prompt diagnosis of dengue. Methods We have demonstrated the development of a magneto-enzyme LFIA combining super-paramagnetic nanoparticles as labels and Biotin–Streptavidin signal amplification strategy to detect dengue NS1. Factors affecting the test performance including antibody pair, super-paramagnetic nanoparticle size, nitrocellulose membrane type, amounts of detection and capture antibodies, and amounts of Streptavidin-polyHRP were optimized. Analytical sensitivity and cross-reactivity were determined. Clinical performance of the novel assay was evaluated using a panel of 120 clinical sera. Results This newly developed assay could detect NS1 of all four serotypes of dengue virus (DENV). The limit of detection (LOD) was found to be as low as 0.25 ng ml−1 for DENV-1 and DENV-3, 0.1 ng ml−1 for DENV-2, and 1.0 ng ml−1 for DENV-4. The LOD for DENV-2 was a 50-fold improvement over the best values previously reported. There was an absence of cross-reactivity with Zika NS1, Hepatitis B virus, Hepatitis C virus, and Japanese encephalitis virus. The sensitivity and specificity of the novel assay were 100% when tested on clinical samples. Conclusions We have successfully developed a magneto-enzyme LFIA, allowing rapid and highly sensitive detection of dengue NS1, which is essential for proper management of patients infected with DENV.

Multiplex PCR and Nanopore Sequencing of Genes Associated with Antimicrobial Resistance in Neisseria gonorrhoeae Directly from Clinical Samples

2020 ◽  
Author(s):  
Chi Zhang ◽  
Leshan Xiu ◽  
Yamei Li ◽  
Liying Sun ◽  
Yizhun Li ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Target Genes ◽  
Limit Of Detection ◽  
Reference Method ◽  
High Sensitivity ◽  
Amplicon Sequencing ◽  
Time Frame ◽  
Cross Reactivity ◽  
Clinical Samples

Abstract Background Antimicrobial resistance (AMR) of Neisseria gonorrhoeae has spread worldwide. Rapid and comprehensive methods are needed to describe N. gonorrhoeae AMR profiles accurately. A method based on multiplex amplicon sequencing was developed to simultaneously sequence 13 genes related to AMR in N. gonorrhoeae directly from clinical samples. Methods Nine N. gonorrhoeae strains were used for the establishment and validation of the method. Eleven urethral swabs and their corresponding cultured isolates were matched as pairs to determine the accuracy of the method. Mock samples with different dilutions were prepared to determine the sensitivity of the method. Five nongonococcal Neisseria strains and 24 N. gonorrhoeae negative clinical samples were used to evaluate the cross-reactivity. Finally, the method was applied to 64 clinical samples to assess its performance. Results Using Sanger sequencing as a reference method, sequences recovered from amplicon sequencing had a base accuracy of over 99.5% and the AMR sites were correctly identified. The limit of detection (LOD) was lower than 31 copies/reaction. No significant cross-reactivity was observed. Furthermore, target genes were successfully recovered from 64 clinical samples including 9 urines, demonstrating this method could be used in different types of samples. For clinical samples, the results can be obtained within a time frame of 7 h 40 min to 10 h 40 min, while for isolates, the turnaround time was approximately 2 h shorter. Conclusions This method can serve as a versatile and convenient culture-free diagnostic method with the advantages of high sensitivity and accuracy.

scholarly journals Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32

2022 ◽  
Vol 16 (1) ◽  
pp. e0010112
Author(s):  
Sirawit Jirawannaporn ◽  
Umaporn Limothai ◽  
Sasipha Tachaboon ◽  
Janejira Dinhuzen ◽  
Patcharakorn Kiatamornrak ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Clinical Performance ◽  
Point Of Care ◽  
Limited Resource ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Detection Assay ◽  
Short Palindromic Repeat ◽  
Flow Detection ◽  
Point Of Care Detection

Background One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples. Methodology/Principal findings A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4–6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study. Conclusions/Significance The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

scholarly journals 422. Performance Evaluation of a Rapid and Easy-to-Use COVID-19 Test

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S278-S278
Author(s):  
Corike Toxopeus ◽  
Brian Jones ◽  
Jessica Brown ◽  
Mark Gurling ◽  
Cynthia Andjelic ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Research Study ◽  
Limit Of Detection ◽  
Scientific Research ◽  
Cross Reactivity ◽  
External Control ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Clinical Specimens ◽  
Study Investigator

Abstract Background The BioFire® COVID-19 Test is a qualitative test for use on the FilmArray® 2.0 and Torch systems for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs (NPS) in transport media. This test received Emergency Use Authorization from the FDA. A closed, disposable pouch contains all the necessary reagents for sample preparation, nucleic acid extraction, reverse transcription, polymerase chain reaction (PCR), and amplified nucleic acid detection to identify RNA from SARS-CoV-2 virus in an NPS specimen. Internal controls monitor all stages of the test process. Once an NPS sample (0.3 mL) is loaded into the system disposable pouch (Figure 1), the fully automated test returns results within an hour. As an additional resource, the BioFire® COVID-19 Test External Control Kit (+) includes positive external control material that may be used for quality control and laboratory verification. Figure 1. BioFire COVID-19 Test Disposable Pouch Methods The following were evaluated: • Limit of Detection (LoD) • Positive and Negative Percent Agreement (PPA and NPA, respectively) for clinical contrived samples and a limited number of clinical specimens • Exclusivity Results • LoD The LoD was evaluated using live SARS-CoV-2 virus (cultured from the USA_WA1/2020 strain obtained from World Reference Center for Emerging Viruses and Arboviruses (WRCEVA)). The LoD was determined to be 3.3E+02 GC/mL (2.2E-02 TCID50/mL). • Clinical Contrived Accurate detection of virus in clinical matrix was demonstrated at various LoD levels using thirty contrived individual unique clinical samples (PPA), and 66 individual unique negative clinical specimens (NPA). • Clinical Samples Positive samples were collected from patients presenting with signs or symptoms of COVID-19, and who were previously identified as positive for SARS-CoV-2 by another EUA test. Negative samples were collected in 2018, and therefore presumed negative for SARS-CoV-2. • Exclusivity The potential for cross-reactivity was evaluated for six viruses from the same genetic family as SARS- CoV-2, and for an additional 30 high priority organisms/viruses. No cross-reactivity was observed. Table 1. SARS-CoV-2 Virus Test Results at 1× and 0.1× LoD for the BioFire COVID-19 Test Table 2. Clinical Contrived and Negative Testing with the BioFire COVID-19 Test Table 3. BioFire COVID-19 Test Performance Summary Conclusion The BioFire COVID-19 Test reliably detects SARS-CoV-2 virus RNA in clinically relevant samples. Disclosures Corike Toxopeus, PhD, BioFire Defense, LLC. (Employee, stock owner) Brian Jones, PhD., BioFire Defense, LLC (Employee, own stock) Jessica Brown, BS, BioFire Defense (Employee, Stock owner) Mark Gurling, PhD, BioFire Defense, LLC (Employee) Cynthia Andjelic, PhD., BioFire Defense (Employee, Other Financial or Material Support, Own stocks) Cynthia L. Phillips, PhD, BioFire Defense (Employee, Scientific Research Study Investigator, Shareholder)BioFire Defense (Employee, Scientific Research Study Investigator, Shareholder)

scholarly journals Accuracy of the Mologic COVID-19 rapid antigen test: a prospective multi-centre analytical and clinical evaluation

2021 ◽  
Vol 6 ◽  
pp. 132
Author(s):  
Ana I Cubas-Atienzar ◽  
Fiona Bell ◽  
Rachel L. Byrne ◽  
Kate Buist ◽  
David J. Clark ◽  
...  
Keyword(s):  
Clinical Presentation ◽  
Limit Of Detection ◽  
Sampling Strategy ◽  
Cross Reactivity ◽  
Analytical Sensitivity ◽  
Antigen Test ◽  
Point Of Use ◽  
Clinical Accuracy ◽  
Polymerase Chain ◽  
Sensitivity Specificity

Background: The coronavirus disease 2019 (COVID-19) pandemic has highlighted the reliance on antigen detection rapid diagnostic tests (Ag-RDTs). Their evaluation at point of use is a priority. Methods: Here, we report a multi-centre evaluation of the analytical sensitivity, specificity, and clinical accuracy of the Mologic COVID-19 Ag-RDT by comparing to reverse transcriptase polymerase chain reaction (RT-qPCR) results from individuals with and without COVID-19 symptoms. Participants had attended hospitals in Merseyside, hospital and ambulance services in Yorkshire, and drive-through testing facilities in Northumberland, UK. Results: The limit of detection of the Mologic COVID-19 Ag-RDT was 5.0 x 102 pfu/ml in swab matrix with no cross-reactivity and interference for any other pathogens tested. A total of 347 participants were enrolled from 26th of November 2020 to 15th of February 2021 with 39.2% (CI 34.0-44.6) testing RT-qPCR positive for SARS-CoV-2. The overall sensitivity and specificity of the Mologic Ag-RDT compared to the reference SARS-CoV-2 RT-qPCR were 85.0% (95% CI 78.3-90.2) and 97.8% (95.0-99.3), respectively. Sensitivity was stratified by RT-qPCR cycle threshold (Ct) and 98.4% (91.3-100) of samples with a Ct less than 20 and 93.2% (86.5-97.2) of samples with a Ct less than 25 were detected using the Ag-RDT. Clinical accuracy was stratified by sampling strategy, swab type and clinical presentation. Mologic COVID-19 Ag-RDT demonstrated highest sensitivity with nose/throat swabs compared with throat or nose swabs alone; however, the differences were not statistically significant. Conclusions: Overall, the Mologic test had high diagnostic accuracy across multiple different settings, different demographics, and on self-collected swab specimens. These findings suggest the Mologic rapid antigen test may be deployed effectively across a range of use settings.

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