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 Performance evaluation of the point-of-care SAMBA II SARS-CoV-2 Test for detection of SARS-CoV-2

2020 ◽  
Author(s):  
Sonny M Assennato ◽  
Allyson V Ritchie ◽  
Cesar Nadala ◽  
Neha Goel ◽  
Hongyi Zhang ◽  
...  
Keyword(s):  
Public Health ◽  
Infection Control ◽  
Sensitivity And Specificity ◽  
Patient Management ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Point Of Care Testing ◽  
Clinical Sensitivity

AbstractNucleic acid amplification for the detection of SARS-CoV-2 RNA in respiratory samples is the standard method for diagnosis. These tests are centralised and therefore turnaround times can be 2-5 days. Point-of-care testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly.Inclusivity and specificity of the SAMBA II SARS-CoV-2 assay was determined by in silico analyses of the primers and probes. Analytical and clinical sensitivity and specificity of the SAMBA II SARS-CoV-2 Test was evaluated for analytical sensitivity and specificity. Clinical performance was evaluated in residual clinical samples compared to the Public Health England reference tests.The limit of detection of the SAMBA II SARS-CoV-2 Test is 250 cp/mL and is specific for detection of 2 regions of the SARS-CoV-2 genome. The clinical sensitivity was evaluated in 172 clinical samples provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge (CMPHL), which showed a sensitivity of 98.9% (95% CI 94.03-99.97%), specificity of 100% (95% CI 95.55-100%), PPV of 100% and NPV of 98.78% (92.02-99.82%) compared to testing by CMPHLSAMBA detected 3 positive samples that were initially negative by PHE Test. The data shows that the SAMBA II SARS-CoV-2 Test performs equivalently to the centralised testing methods with a much quicker turnaround time. Point of care testing, such as SAMBA, should enable rapid patient management and effective implementation of infection control measures.

scholarly journals A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

2021 ◽  
Vol 11 ◽  
Author(s):  
Iqbal Azmi ◽  
Md Imam Faizan ◽  
Rohit Kumar ◽  
Siddharth Raj Yadav ◽  
Nisha Chaudhary ◽  
...  
Keyword(s):  
Data Storage ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Smartphone Application ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Analytical Sensitivity ◽  
Test Results ◽  
Extraction Step

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13AssistedSaliva-based &SmartphoneIntegratedTesting), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.

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 Semi-Quantitative, Duplexed qPCR Assay for the Detection of Leishmania spp. Using Bisulphite Conversion Technology

2019 ◽  
Vol 4 (4) ◽  
pp. 135 ◽  
Author(s):  
Ineka Gow ◽  
Douglas Millar ◽  
John Ellis ◽  
John Melki ◽  
Damien Stark
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
World Health ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Internal Transcribed Spacer Region ◽  
Leishmania Spp ◽  
Sensitivity Data ◽  
Conversion Technology

Leishmaniasis is caused by the flagellated protozoan Leishmania, and is a neglected tropical disease (NTD), as defined by the World Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all Leishmania species. The assay was validated against previously extracted DNA, from seven quantitated DNA and cell standards for pan-Leishmania analytical sensitivity data, and 67 cutaneous clinical samples for cutaneous clinical sensitivity data. Specificity was evaluated by testing 76 negative clinical samples and 43 bacterial, viral, protozoan and fungal species. The assay was also trialed in a side-by-side experiment against a conventional PCR (cPCR), based on the Internal transcribed spacer region 1 (ITS1 region). Ninety-seven percent of specimens from patients that previously tested positive for Leishmania were positive for Leishmania spp. with the bisulphite conversion assay, and a limit of detection (LOD) of 10 copies per PCR was achieved, while the LOD of the ITS1 methodology was 10 cells/1000 genomic copies per PCR. This method of rapid, accurate and simple detection of Leishmania can lead to improved diagnosis, treatment and public health outcomes.

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 Pathogenic and Virulence Factor Detection on Viable but Non-culturable Methicillin-Resistant Staphylococcus aureus

2021 ◽  
Vol 12 ◽  
Author(s):  
Hua Jiang ◽  
Kan Wang ◽  
Muxia Yan ◽  
Qian Ye ◽  
Xiaojing Lin ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Limit Of Detection ◽  
Foodborne Pathogen ◽  
Food Poisoning ◽  
Clinical Settings ◽  
Analytical Sensitivity ◽  
Vbnc State ◽  
Methicillin Resistant ◽  
Staphylococcal Enterotoxins ◽  
Sensitivity Specificity

Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called “standards,” “guidelines,” or “gold standards” are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/μl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.

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.

scholarly journals Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis

2020 ◽  
Vol 9 (1) ◽  
pp. 41
Author(s):  
Veronika Pilchová ◽  
Diana Seinige ◽  
Isabel Hennig-Pauka ◽  
Kathrin Büttner ◽  
Amir Abdulmawjood ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Glaesserella parasuis is a fastidious pathogen that colonizes the respiratory tract of pigs and can lead to considerable economic losses in pig production. Therefore, a rapid detection assay for the pathogen, preferably applicable in the field, is important. In the current study, we developed a new and improved detection method using loop-mediated isothermal amplification (LAMP). This assay, which targets the infB gene, was tested on a collection of 60 field isolates of G. parasuis comprising 14 different serovars. In addition, 63 isolates from seven different closely related species of the family Pasteurellaceae, including A. indolicus, A. porcinus, and A. minor, and a species frequently found in the respiratory tract of pigs were used for exclusivity experiments. This assay showed an analytical specificity of 100% (both inclusivity and exclusivity) and an analytical sensitivity of 10 fg/µL. In further steps, 36 clinical samples were tested with the LAMP assay. An agreement of 77.1 (95% CI: 59.9, 89.6) and 91.4% (95% CI: 75.9, 98.2) to the culture-based and PCR results was achieved. The mean limit of detection for the spiked bronchoalveolar lavage fluid was 2.58 × 102 CFU/mL. A colorimetric assay with visual detection by the naked eye was tested to provide an alternative method in the field and showed the same sensitivity as the fluorescence-based LAMP assay. Overall, the optimized LAMP assay represents a fast and reliable method and is suitable for detecting G. parasuis in the laboratory environment or in the field.

scholarly journals Development and validation of a multigene variant profiling assay to guide targeted and immuno therapy selection in solid tumors

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246048
Author(s):  
Dadasaheb Akolkar ◽  
Darshana Patil ◽  
Navin Srivastava ◽  
Revati Patil ◽  
Vineet Datta ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Clinical Performance ◽  
High Specificity ◽  
Clinical Samples ◽  
Solid Organ ◽  
Analytical Sensitivity ◽  
Mutation Burden ◽  
Validation Parameters ◽  
Sensitivity Specificity ◽  
Selection Of

We present data on analytical validation of the multigene variant profiling assay (CellDx) to provide actionable indications for selection of targeted and immune checkpoint inhibitor (ICI) therapy in solid tumors. CellDx includes Next Generation Sequencing (NGS) profiling of gene variants in a targeted 452-gene panel as well as status of total Tumor Mutation Burden (TMB), Microsatellite instability (MSI), Mismatch Repair (MMR) and Programmed Cell Death—Ligand 1 (PD-L1) respectively. Validation parameters included accuracy, sensitivity, specificity and reproducibility for detection of Single Nucleotide Alterations (SNAs), Copy Number Alterations (CNAs), Insertions and Deletions (Indels), Gene fusions, MSI and PDL1. Cumulative analytical sensitivity and specificity of the assay were 99.03 (95% CI: 96.54–99.88) and 99.23% (95% CI: 98.54% - 99.65%) respectively with 99.20% overall Accuracy (95% CI: 98.57% - 99.60%) and 99.7% Precision based on evaluation of 116 reference samples. The clinical performance of CellDx was evaluated in a subsequent analysis of 299 clinical samples where 861 unique mutations were detected of which 791 were oncogenic and 47 were actionable. Indications in MMR, MSI and TMB for selection of ICI therapies were also detected in the clinical samples. The high specificity, sensitivity, accuracy and reproducibility of the CellDx assay is suitable for clinical application for guiding selection of targeted and immunotherapy agents in patients with solid organ tumors.

scholarly journals Approbation of RT-qPCR test kit for differential diagnosis of African and Classical swine fever

2018 ◽  
Vol 20 (83) ◽  
pp. 221-225
Author(s):  
S.S. Mandyhra ◽  
L.M. Muzykina ◽  
L.M. Ishchenko ◽  
G.A. Kovalenko ◽  
I.V. Halka ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
Veterinary Medicine ◽  
Internal Control ◽  
Limit Of Detection ◽  
Control Sample ◽  
Classical Swine Fever ◽  
Analytical Sensitivity ◽  
Test Kit ◽  
Field Samples ◽  
Sensitivity Specificity

The first Ukrainian real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) based test kit for the differential diagnosis of African (AFS) and Classical swine fever (CSF) has been developed in the Institute of Veterinary Medicine of NAAS. The proposed test kit allows simultaneous detection of three targets: ASFV DNA, CSFV cDNA and an internal control sample. The goal of this work was to provide an expert evaluation of the RT-qPCR kit for differential diagnosis of ASF and CSF according to appearance, analytical sensitivity, specificity and repeatability. Interdepartmental evaluation of the kit was conducted in the State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (DNDILDEVSE) in accordance with the approved methodology. The RT-qPCR kit sensitivity was determined by testing 10-fold serial dilutions of the ASFV DNA and CSFV cDNA (concentration range was 103–100 copies/μl). For specificity determination reference samples of ASFV DNA different genotypes, ASF and CSF positive and negative field samples, as well as pathogens which cause similar to ASF and CSF clinical syndromes were used. Sample preparation and amplification were performed according to the test kit instructions. The amplification was accomplished on QuantStudio™ 5 System (Applied Biosystems). As a result of accomplished interdepartmental evaluation high sensitivity, specificity and repeatability of RT-qPCR kit were confirmed. In particular, it was determined that the limit of detection of the RT-qPCR kit was 5 copies of the ASFV and CSFV genomes per one reaction. The high specificity of the assay to ASFV (I, II, V, VIII, IX and X genotypes) and CSFV was confirmed. It was showп no cross-reactions with closely related pigs viruses (porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and virus of Aujeszky's disease). The high enough repeatability of results was also confirmed. In conclusion, the obtained results are in compliance with the requirements of the RT-qPCR kit normative and technical documentation. This RT-qPCR kit will be recommended for use in veterinary medicine laboratories after its registration would be done.

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