Spore immobilization and its analytical performance for monitoring of aflatoxin M1 in milk

2014 ◽  
Vol 60 (12) ◽  
pp. 793-798
Author(s):  
V.K. Singh ◽  
N.A. Singh ◽  
N. Kumar ◽  
H.V. Raghu ◽  
Pradeep Kumar Sharma ◽  
...  
Keyword(s):  
Real Time ◽  
False Positive ◽  
Physical Adsorption ◽  
False Negative ◽  
Colour Change ◽  
Dairy Farm ◽  
Cost Effective ◽  
Visual Observation ◽  
Detection Sensitivity ◽  
Aflatoxin M1

Immobilization of Bacillus megaterium spores on Eppendorf tubes through physical adsorption has been used in the detection of aflatoxin M1 (AFM1) in milk within real time of 45 ± 5 min using visual observation of changes in a chromogenic substrate. The appearance of a sky-blue colour indicates the absence of AFM1 in milk, whereas no colour change indicates the presence of AFM1 in milk at a 0.5 ppb Codex maximum residue limit. The working performance of the immobilized spores was shown to persist for up to 6 months. Further, spores immobilized on 96-well black microtitre plates by physical adsorption and by entrapment on sensor disk showed a reduction in detection sensitivity to 0.25 ppb within a time period of 20 ± 5 min by measuring fluorescence using a microbiological plate reader through the addition of milk and fluorogenic substrate. A high fluorescence ratio indicated more substrate hydrolysis due to spore-germination-mediated release of marker enzymes of spores in the absence of AFM1 in milk; however, low fluorescence ratios indicated the presence of AFM1 at 0.25 ppb. Immobilized spores on 96-well microtitre plates and sensor disks have shown better reproducibility after storage at 4 °C for 6 months. Chromogenic assay showed 1.38% false-negative and 2.77% false-positive results while fluorogenic assay showed 4.16% false-positive and 2.77% false-negative results when analysed for AFM1 using 72 milk samples containing raw, pasteurized, and dried milk. Immobilization of spores makes these chromogenic and fluorogenic assays portable, selective, cost-effective for real-time detection of AFM1 in milk at the dairy farm, reception dock, and manufacturing units of the dairy industry.

scholarly journals Proposal of De Novo Antigen Test for COVID-19: Ultrasensitive Detection of Spike Proteins of SARS-CoV-2

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 594 ◽  
Author(s):  
Yuta Kyosei ◽  
Mayuri Namba ◽  
Sou Yamura ◽  
Rikiya Takeuchi ◽  
Noriko Aoki ◽  
...  
Keyword(s):  
De Novo ◽  
Limit Of Detection ◽  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Test ◽  
Virus Rna ◽  
Antigen Tests ◽  
Spike Proteins

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10−18 moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10−20 moles of virus/assay, corresponding to ~104 copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~105 copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19.

scholarly journals False positive antigen test for Dirofilaria immitis after heat treatment of the blood sample in a microfilaremic dog infected with Acanthocheilonema dracunculoides

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Viktor Szatmári ◽  
Martin Willem van Leeuwen ◽  
Christine Jantine Piek ◽  
Luigi Venco
Keyword(s):  
Heat Treatment ◽  
Blood Sample ◽  
Real Time ◽  
Real Time Pcr ◽  
False Positive ◽  
Dirofilaria Immitis ◽  
False Negative ◽  
Correct Identification ◽  
Test Results ◽  
Antigen Test

Abstract Background Dirofilaria immitis is responsible for heartworm disease in dogs in endemic areas worldwide. Screening for this infection is done by blood tests. Antigen testing is the most sensitive method to detect an infection with adult (female) worms. Microscopic examination of a blood smear or Knott’s test can be used to detect circulating microfilariae, the infective larvae. To increase the sensitivity of the antigen test by decreasing the false negative test results, heating of the blood sample has been recommended in recent guidelines. Heating is believed to remove blocking immune-complexes. Circulating microfilariae are not specific findings for heartworm infection, as other nematodes (among others, Acanthocheilonema dracunculoides) can also result in microfilaremia. Although the type of microfilariae cannot be determined by microscopy alone, real-time PCR can reliably identify the infecting nematode species. Correct identification of the parasite is of major importance, as an infection with D. immitis requires antiparasitic therapy, whereas A. dracunculoides is thought to be a clinically irrelevant coincidental finding. The present case report describes a microfilaremic dog where the initial antigen test for D. immitis turned positive after heat treatment, whereas real-time PCR revealed that the microfilariae were A. dracunculoides (syn. Dipetalonema dracunculoides). Results A circa 5-year old, asymptomatic Spanish mastiff dog was referred for heartworm therapy because microfilariae were found via a screening blood test. The dog was recently imported to the Netherlands from Spain, where it had been a stray dog. Antigen tests on a plasma sample for D. immitis were performed with three different test kits, which all turned out to be negative. However, heat treatment of two of these samples were carried out and both of them led to a positive antigen test result. Real-time PCR showed that the circulating microfilariae belonged to A. dracunculoides species. Three administrations of moxidectin spot-on at monthly intervals resulted in a negative antigen and a negative Knott’s tests one month after the last treatment. Conclusions We conclude that heat treatment of initially negative blood samples for D. immitis could lead to false positive antigen test results if the dog is infected with A. dracunculoides.

scholarly journals Shine: To explore specific, sensitive and conserved biomarkers from massive microbial genomic data within intrapopulations.

2021 ◽  
Author(s):  
Cong Ji ◽  
Junbin Jack Shao
Keyword(s):  
False Negative ◽  
Genomic Data ◽  
Cost Effective ◽  
Pcr Primers ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Pathogenic Microorganisms ◽  
New Strategy ◽  
Microbial Genomic ◽  
Species Specific

To improve the quality of nucleic acid detection reagents, we provided a new strategy, Shine, to explore specific, sensitive and conserved biomarkers from massive microbial genomic data within intrapopulations in order to improve detection sensitivity and accuracy. It is obvious that the more comprehensive genomic data are, the more effective the detection biomarkers. Here, we demonstrated that our method could detect undiscovered multicopy conserved species-specific or even subspecies-specific target fragments, according to several clinical projects. In particular, this approach was effective for any pathogenic microorganism even in incompletely assembled motifs. Based on our strategy, the detection device designed with quantitative PCR primers and probes for systematic and automated detection of pathogenic microorganisms in biological samples may cover all pathogenic microorganisms without limits based on genome annotation. On the website https://bioinfo.liferiver.com.cn, users may select different configuration parameters depending on the purpose of the project to realize routine clinical detection practices. Therefore, it is recommended that our strategy is suitable to identify shared universal phylogenetic markers with few false positive or false negative errors and to automate the design of minimal primers and probes to detect pathogenic communities with cost-effective predictive power.

Accuracy of Fine-Needle Aspiration of Thyroid

2001 ◽  
Vol 125 (4) ◽  
pp. 484-488 ◽  
Author(s):  
Mojghan Amrikachi ◽  
Ibrahim Ramzy ◽  
Sheldon Rubenfeld ◽  
Thomas M. Wheeler
Keyword(s):  
Fine Needle Aspiration ◽  
False Positive ◽  
Thyroid Nodules ◽  
False Negative ◽  
Cost Effective ◽  
Needle Aspiration ◽  
Follicular Neoplasm ◽  
Follicular Variant ◽  
Fine Needle

Abstract Context.—Fine-needle aspiration has become an accepted and cost-effective procedure for rapid diagnosis of thyroid lesions. The routine use of fine-needle aspiration has reduced the rate of unnecessary surgery for thyroid nodules. Objectives.—To determine the accuracy of fine-needle aspiration biopsy diagnosis and to discuss the possible pitfalls. Design, Setting, and Participants.—Reports of 6226 fine-needle aspiration biopsies of the thyroid performed during a period of 16 years (1982–1998) were reviewed. Computerized reports of the fine-needle aspiration biopsies were sent to the physicians who performed the procedures, and clinical follow-up information regarding the patients was requested. Twenty-four clinicians participated in the study. Histologic diagnoses were available for 354 cases. The cytopathologic diagnoses were correlated with the histologic findings or clinical outcomes. Results.—The cytologic diagnoses were as follows: 210 (3.4%) malignant, 450 (7.2%) suspicious, 3731 (60%) benign, and 1845 (29.5%) unsatisfactory. Most of the cases with negative or unsatisfactory aspirates were followed clinically or by repeat fine-needle aspiration. We identified 11 false-negative and 7 false-positive diagnoses. For aspirates considered sufficient for diagnosis, the sensitivity and specificity levels were 93% and 96%, respectively. Conclusions.—Fine-needle aspiration of the thyroid gland is highly accurate and has a low rate of false-negative and false-positive diagnoses. The major diagnostic problems are caused by diagnosis using a marginally adequate specimen, diagnosis of malignancy based on just 1 or 2 atypical cytologic features, or overlapping cytologic features of follicular neoplasm with those of follicular variant of papillary carcinoma.

Comparison of real-time PCR with fungal culture for the diagnosis of Microsporum canis dermatophytosis in shelter cats: a field study

2017 ◽  
Vol 20 (2) ◽  
pp. 103-107 ◽  
Author(s):  
Linda S Jacobson ◽  
Lauren McIntyre ◽  
Jenny Mykusz
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
False Positive ◽  
False Negative ◽  
Microsporum Canis ◽  
Fungal Culture ◽  
Mycological Cure ◽  
Hair Samples ◽  
Culture Positive ◽  
Culture Negative

Objectives Fungal culture requires at least 14 days for a final result, compared with 1–3 days for PCR. The study compared a commercial real-time dermatophyte PCR panel with fungal culture in cats in a shelter setting for: (1) diagnosis of Microsporum canis infection; and (2) determination of mycological cure. Methods This was a cross-sectional, observational study of cats with suspicious skin lesions or suspected exposure to dermatophytosis. Hair samples were collected for fungal culture and PCR prior to treatment and at weekly intervals until two negative culture results were obtained. Results One hundred and thirty-two cats were included, of which 28 (21.2%) were culture positive and 104 (78.8%) culture-negative for M canis. PCR correctly identified all culture-positive cats and 92/104 culture negative cats; there were 12 false-positive PCR results. The sensitivity and specificity of PCR were 100% (95% confidence interval [CI] 87.7–100) and 88.5% (95% CI 80.7–93.9), respectively. Data from 17 cats were available for assessment of mycological cure. At the time of the first and second negative fungal cultures, 14/17 (82.4%) and 11/17 (64.7%) tested PCR positive, respectively. Conclusions and relevance PCR showed high sensitivity and specificity for diagnosis of M canis dermatophytosis compared with fungal culture, but was unreliable for identifying mycological cure. False-positive results were relatively common. There were no false-negative PCR results and a negative PCR test was a reliable finding in this study. The ability to rapidly diagnose or rule out dermatophytosis could be a valuable tool to increase life-saving capacity in animal shelters.

scholarly journals IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

2020 ◽  
Author(s):  
Elizabeth C. Stahl ◽  
Connor A. Tsuchida ◽  
Jennifer R. Hamilton ◽  
Enrique Lin-Shiao ◽  
Shana L. McDevitt ◽  
...  
Keyword(s):  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
N Gene ◽  
Negative Results ◽  
False Negative Results ◽  
Single Well ◽  
Sample Pooling ◽  
One Step ◽  
Viral Target

AbstractCommonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.Graphical Abstract

scholarly journals Comparison of real-time PCR and droplet digital PCR for the detection of Xylella fastidiosa in plants

2019 ◽  
Author(s):  
Enora Dupas ◽  
Bruno Legendre ◽  
Valérie Olivier ◽  
Françoise Poliakoff ◽  
Charles Manceau ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Xylella Fastidiosa ◽  
False Negative ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Detection Sensitivity ◽  
Plant Sample ◽  
The European Union

AbstractXylella fastidiosa (Xf) is a quarantine plant pathogen bacterium originating from the Americas and that has emerged in Europe in 2013. Xf can be detected directly on plant macerate using molecular methods such as real-time PCR, which is a sensitive technique. However, some plants may contain components that can act as PCR reaction inhibitors, which can lead to false negative results or an underestimation of the bacterial concentration present in the analyzed plant sample. Droplet digital PCR (ddPCR) is an innovative tool based on the partitioning of the PCR reagents and the DNA sample into thousands of droplets, allowing the quantification of the absolute number of target DNA molecules present in a reaction mixture, or an increase of the detection sensitivity. In this study, a real-time PCR protocol, already used for Xf detection in the framework of official surveys in the European Union, was transferred and optimized for Xf detection using ddPCR. This new assay was evaluated and compared to the initial real-time PCR on five plant matrices artificially inoculated and on naturally infected plants. In our conditions, this new ddPCR enabled the detection of Xf on all artificially inoculated plant macerates with a similar limit of detection, or a slight benefit for Quercus ilex. Moreover, ddPCR improved diagnostic sensitivity as it enabled detection of Xf in samples of Polygala myrtifolia or Q. ilex that were categorized as negative or close to the limit of detection using the real-time PCR. Here, we report for the first time a ddPCR assay for the detection of the bacterium Xf.

scholarly journals Cost-effectiveness of WHO-Recommended Algorithms for TB Case Finding at Ethiopian HIV Clinics

2017 ◽  
Vol 5 (1) ◽  
Author(s):  
Max W Adelman ◽  
Deborah A McFarland ◽  
Mulugeta Tsegaye ◽  
Abraham Aseffa ◽  
Russell R Kempker ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
False Positive ◽  
False Negative ◽  
Cost Effective ◽  
Case Finding ◽  
World Health ◽  
Molecular Diagnostic ◽  
True Positive ◽  
High Burden ◽  
Model Based

Abstract Background The World Health Organization (WHO) recommends active tuberculosis (TB) case finding and a rapid molecular diagnostic test (Xpert MTB/RIF) to detect TB among people living with HIV (PLHIV) in high-burden settings. Information on the cost-effectiveness of these recommended strategies is crucial for their implementation. Methods We conducted a model-based cost-effectiveness analysis comparing 2 algorithms for TB screening and diagnosis at Ethiopian HIV clinics: (1) WHO-recommended symptom screen combined with Xpert for PLHIV with a positive symptom screen and (2) current recommended practice algorithm (CRPA; based on symptom screening, smear microscopy, and clinical TB diagnosis). Our primary outcome was US$ per disability-adjusted life-year (DALY) averted. Secondary outcomes were additional true-positive diagnoses, and false-negative and false-positive diagnoses averted. Results Compared with CRPA, combining a WHO-recommended symptom screen with Xpert was highly cost-effective (incremental cost of $5 per DALY averted). Among a cohort of 15 000 PLHIV with a TB prevalence of 6% (900 TB cases), this algorithm detected 8 more true-positive cases than CRPA, and averted 2045 false-positive and 8 false-negative diagnoses compared with CRPA. The WHO-recommended algorithm was marginally costlier ($240 000) than CRPA ($239 000). In sensitivity analysis, the symptom screen/Xpert algorithm was dominated at low Xpert sensitivity (66%). Conclusions In this model-based analysis, combining a WHO-recommended symptom screen with Xpert for TB diagnosis among PLHIV was highly cost-effective ($5 per DALY averted) and more sensitive than CRPA in a high-burden, resource-limited setting.

scholarly journals LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0258263
Author(s):  
Elizabeth C. Stahl ◽  
Allan R. Gopez ◽  
Connor A. Tsuchida ◽  
Vinson B. Fan ◽  
Erica A. Moehle ◽  
...  
Keyword(s):  
Large Scale ◽  
Local Community ◽  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
N Gene ◽  
Negative Results ◽  
Clinical Surveillance ◽  
Sample Pooling ◽  
Wastewater Samples

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

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