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.

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 A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, speciation, and drug-resistance genotyping

2020 ◽  
Author(s):  
CH Cunningham ◽  
CM Hennelly ◽  
JT Lin ◽  
R Ubalee ◽  
RM Boyce ◽  
...  
Keyword(s):  
False Negative ◽  
High Sensitivity ◽  
Disease Diagnosis ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Robust Detection ◽  
Drug Resistance Genotyping ◽  
Republic Of The Congo ◽  
Species Specific ◽  
Analytical Sensitivities

ABSTRACTCRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection. We developed SHERLOCK assays for malaria capable of detecting all Plasmodium species known to cause malaria in humans and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We validated these assays against parasite genomic DNA and achieved analytical sensitivities ranging from 2.5-18.8 parasites per reaction. We further tested these assays using a diverse panel of 123 clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity in clinical samples. In addition, we developed a SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine-pyrimethamine resistance. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities. These novel SHERLOCK assays have potential to spawn a new generation of molecular diagnostics for malaria and demonstrate the versatility of CRISPR-based diagnostic approaches.One-sentence summaryNovel malaria SHERLOCK assays enabled robust detection, speciation, and genotyping of Plasmodium spp. in diverse samples collected in Africa and Asia.

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

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 Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241469
Author(s):  
Yunying Zhou ◽  
Fengyan Pei ◽  
Mingyu Ji ◽  
Li Wang ◽  
Huailong Zhao ◽  
...  
Keyword(s):  
Viral Load ◽  
Nucleic Acid ◽  
False Negative ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Current Standard ◽  
Mixed Sample ◽  
Asymptomatic Patients ◽  
Selection Of

The early detection and differential diagnosis of respiratory infections increase the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis. However, the maximal specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, recent evidence indicated that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” might be due to low viral load, and the ability of rapid mutation of SARS-CoV-2 also increases the rate of false-negative results. Moreover, the mixed sample nucleic acid detection is helpful in seeking out the early community transmission of SARS-CoV-2 rapidly, but the detection kit needs ultra-high detection sensitivity. Herein, the lowest detection concentration of different nucleic acid detection kits was evaluated and compared to provide direct evidence for the selection of kits for mixed sample detection or make recommendations for the selection of validation kit, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.

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.

scholarly journals Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts

2008 ◽  
Vol 53 (No. 3) ◽  
pp. 97-104 ◽  
Author(s):  
M. Zouhar ◽  
M. Marek ◽  
O. Douda ◽  
J. Mazáková ◽  
P. Ryšánek
Keyword(s):  
Cost Effective ◽  
Healthy Plant ◽  
Host Preferences ◽  
Sequence Characterized Amplified Region ◽  
Detection And Identification ◽  
Plant Hosts ◽  
Cost Effective Technique ◽  
Ditylenchus Dipsaci ◽  
Species Specific ◽  
Template Dna

<i>Ditylenchus dipsaci</i>, the stem nematode, is a migratory endoparasite of over 500 species of angiosperms. The main method of <i>D. dipsaci</i> control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation generally ineffective. Therefore, a sensitive, rapid, reliable, as well as cost effective technique is needed for identification of <i>D. dipsaci</i> in biological samples. This study describes the development of species-specific pairs of PCR oligonucleotides for detection and identification of the <i>D. dipsaci</i> stem nematode in various plant hosts. Designed DIT-2 primer pair specifically amplified a fragment of 325 bp, while DIT-5 primer pair always produced a fragment of 245 bp in all <i>D. dipsaci</i> isolates. Two developed SCAR primer pairs were further tested using template DNA extracted from a collection of twelve healthy plant hosts; no amplification was however observed. The developed PCR protocol has proved to be quite sensitive and able to specifically detect <i>D. dipsaci</i> in artificially infested plant tissues.

scholarly journals Comparison of saliva with healthcare workers- and patient-collected swabs in the diagnosis of COVID-19 in a large cohort

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mitnala Sasikala ◽  
Yelamanchili Sadhana ◽  
Ketavarapu Vijayasarathy ◽  
Anand Gupta ◽  
Sarala Kumari Daram ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Early Stage ◽  
Cost Effective ◽  
Hospitalized Patients ◽  
Detection Sensitivity ◽  
Nasopharyngeal Swab ◽  
Predictive Values ◽  
Qrt Pcr ◽  
Asymptomatic Patients ◽  
Sensitivity Specificity

Abstract Background A considerable amount of evidence demonstrates the potential of saliva in the diagnosis of COVID-19. Our aim was to determine the sensitivity of saliva versus swabs collected by healthcare workers (HCWs) and patients themselves to assess whether saliva detection can be offered as a cost-effective, risk-free method of SARS-CoV-2 detection. Methods This study was conducted in a hospital involving outpatients and hospitalized patients. A total of 3018 outpatients were tested. Of these, 200 qRT-PCR-confirmed SARS-CoV-2-positive patients were recruited for further study. In addition, 101 SARS-CoV-2-positive hospitalized patients with symptoms were also enrolled in the study. From outpatients, HCWs collected nasopharyngeal swabs (NPS), saliva were obtained. From inpatients, HCWs collected swabs, patient-collected swabs, and saliva were obtained. qRT-PCR was performed to detect SARS-CoV-2 by TAQPATH assay to determine the sensitivity of saliva detection. Sensitivity, specificity and positive/negative predictive values (PPV, NPV) of detecting SARS-CoV-2 were calculated using MedCalc. Results Of 3018 outpatients (asymptomatic: 2683, symptomatic: 335) tested by qRT-PCR, 200 were positive (males: 140, females: 60; aged 37.9 ± 12.8 years; (81 asymptomatic, 119 symptomatic). Of these, saliva was positive in 128 (64%); 39 of 81 asymptomatic (47%),89 of 119 symptomatic patients (74.8%). Sensitivity of detection was 60.9% (55.4–66.3%, CI 95%), with a negative predictive value of 36%(32.9–39.2%, CI 95%).Among 101 hospitalized patients (males:65, females: 36; aged 53.48 ± 15.6 years), with HCW collected NPS as comparator, sensitivity of saliva was 56.1% (47.5–64.5, CI 95%), specificity 63.5%(50.4–75.3, CI95%) with PPV of 77.2% and NPV of 39.6% and that of self-swab was 52.3%(44–60.5%, CI95%), specificity 56.6% (42.3–70.2%, CI95%) with PPV 77.2% and NPV29.7%. Comparison of positivity with the onset of symptoms revealed highest detection in saliva on day 3 after onset of symptoms. Additionally, only saliva was positive in 13 (12.8%) hospitalized patients. Conclusion Saliva which is easier to collect than nasopharyngeal swab is a viable alternate to detect SARS-COV-2 in symptomatic patients in the early stage of onset of symptoms. Although saliva is currently not recommended for screening asymptomatic patients, optimization of collection and uniform timing of sampling might improve the sensitivity enabling its use as a screening tool at community level.

scholarly journals NESTROFT—A Cost-Effective Mass Screening Tool for the Detection of β-Thalassemia Carrier Status in Anemic Pregnant Women: A Step Toward Reducing the National Disease Burden

2021 ◽  
Author(s):  
Manasi Gosavi ◽  
Ramesh Chavan ◽  
M. B. Bellad
Keyword(s):  
Pregnant Women ◽  
Effective Mass ◽  
Mass Screening ◽  
Predictive Value ◽  
Screening Tool ◽  
Hematological Parameters ◽  
False Negative ◽  
Cost Effective ◽  
Mean Corpuscular Hemoglobin ◽  
Carrier Status

Abstract Introduction β-Thalassemias are inherited hemoglobinopathies commonly encountered in practice. With chances of a promising cure being rare, the prevention of births with this disorder should assume priority, especially in low-resource countries. This can be achieved by the implementation of a mass screening program that is reliable and, at the same time, cost-effective. Objectives This study focuses on the utility of Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) as a mass screening tool to detect thalassemia carriers. Hematological parameters that may predict carrier status were also evaluated. Materials and Methods Hemoglobin estimation was performed on all consented pregnant women. If the patient was found to have hemoglobin < 11 g/dL, the blood sample was subjected to other routine hematological tests along with peripheral smear examination. NESTROFT was performed using 0.36% saline solution. Confirmation was done using high-performance liquid chromatography (HPLC). Statistical Analysis Data obtained were tabulated using version 21 of the Statistical Package for Social Sciences. Means, standard deviations, and percentages were used to describe the sample. Chi-square test and Students’ “t” test were used to identify differences between the groups. Results Of 441 pregnant women enrolled, 206 were found to be anemic. Nineteen (9.2%) of the anemic pregnant women were detected to be carriers of hemoglobinopathies. Among the hematological parameters, mean red blood cell count and reticulocyte count were higher, while mean corpuscular hemoglobin concentration was lower in carriers. Also, carriers were more likely to present with microcytic hypochromic anemia. NESTROFT showed a sensitivity of 84.21%, specificity of 96.25%, a positive predictive value of 69.56%, and a negative predictive value of 98.36%. A false-positive result was seen in 3.74% of the tests, while a false negative result was seen in 15.78% of the tests. Conclusions NESTROFT (0.36%) can be used as a simple and cost-effective mass screening tool for the detection of carrier status. This should be followed by confirmation using HPLC or hemoglobin electrophoresis.

A Multiplex PCR Assay Mediated by Universal Primers for the Detection of Adulterated Meat in Mutton

2019 ◽  
Vol 82 (2) ◽  
pp. 325-330 ◽  
Author(s):  
WANWAN LIU ◽  
XIAONAN WANG ◽  
JING TAO ◽  
BANGSHENG XI ◽  
MAN XUE ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Detection System ◽  
Meat Products ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Food Samples ◽  
Detection Sensitivity ◽  
Universal Primers ◽  
Multiplex Pcr Assay ◽  
Multiple Pcr

ABSTRACT This study aimed to establish a multiplex PCR detection system mediated by “universal primers,” which would be able to determine whether mutton meat contained nonmutton ingredients from rats, foxes, and ducks. Based on the sequence variation of specific mitochondrial genes, nine different multiplex PCR primers were designed, and four kinds of meat products were rapidly identified by electrophoresis using an optimized multiplex PCR system based on the molecular weight differences of the amplified products. Multiplex PCR applications optimized for meat food source from food samples for testing was used to verify the accuracy of the identification method. The results showed that the primers in multiple PCR system mediated by universal primers could be used for the rapid identification of rat, fox, duck, and sheep meat in mutton products, and the detection sensitivity could reach 0.05 ng/μL. The identification of food samples validated the practical value of this method. Therefore, a multiplex PCR system mediated by universal primers was established, which can be used to quickly identify the origin of animal ingredients from rats, foxes, and ducks in mutton products.

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