scholarly journals Competitive SNP-LAMP probes for rapid and robust single-nucleotide polymorphism detection

2021 ◽  
Author(s):  
Leland B Hyman ◽  
Clare R Christopher ◽  
Philip A Romero
Keyword(s):  
Point Of Care ◽  
High Specificity ◽  
Common Source ◽  
Lamp Method ◽  
Universal Method ◽  
Nucleotide Polymorphisms ◽  
One Pot ◽  
Single Nucleotide ◽  
Specificity And Sensitivity ◽  
And Shifts

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are typically detected using DNA sequencing, which requires advanced sample preparation and instrumentation, and thus cannot be deployed for on-site testing or in low-resource settings. In this work we have developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines LAMP-based target amplification with fluorescent probes to detect SNPs with high specificity in a one-pot reaction format. A competitive "sink" strand preferentially binds to off-target products and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible SARS-CoV-2 variants. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity.

scholarly journals Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1207
Author(s):  
Hong Jae Cheon ◽  
Quynh Huong Nguyen ◽  
Moon Il Kim
Keyword(s):  
Magnetic Nanoparticles ◽  
High Sensitivity ◽  
High Specificity ◽  
Choline Oxidase ◽  
Fluorescent Detection ◽  
One Pot ◽  
Site Structure ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Peroxidase Mimics

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

scholarly journals Museum Genomics Illuminate the High Specificity of a Bioluminescent Symbiosis for a Genus of Reef Fish

2021 ◽  
Vol 9 ◽  
Author(s):  
Alison L. Gould ◽  
Allison Fritts-Penniman ◽  
Ana Gaisiner
Keyword(s):  
High Specificity ◽  
Bacterial Symbiont ◽  
Light Organ ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
New Approach ◽  
Host Genus ◽  
Symbiotic Relationships ◽  
Luminous Bacteria ◽  
Formalin Fixed

Symbiotic relationships between bioluminescent bacteria and fishes have evolved multiple times across hundreds of fish taxa, but relatively little is known about the specificity of these associations and how stable they are over host generations. This study describes the degree of specificity of a bioluminescent symbiosis between cardinalfishes in the genus Siphamia and luminous bacteria in the Vibrio family. Primarily using museum specimens, we investigated the codivergence of host and symbiont and test for patterns of divergence that correlate with both biogeography and time. Contrary to expectations, we determined that the light organ symbionts of all 14 Siphamia species examined belong to one genetic clade of Photobacterium mandapamensis (Clade II), indicating that the association is highly specific and conserved throughout the host genus. Thus, we did not find evidence of codivergence among hosts and symbionts. We did observe that symbionts hosted by individuals sampled from colder water regions were more divergent, containing more than three times as many single nucleotide polymorphisms than the rest of the symbionts examined. Overall, our findings indicate that the symbiosis between Siphamia fishes and P. mandapamensis Clade II has been highly conserved across host taxa and over a broad geographic range despite the facultative nature of the bacterial symbiont. We also present a new approach to simultaneously recover genetic information from a bacterial symbiont and its vertebrate host from formalin-fixed specimens, enhancing the utility of museum collections.

scholarly journals A rapid and affordable point of care test for antibodies against SARS-CoV-2 based on hemagglutination and artificial intelligence interpretation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vanessa Redecke ◽  
Kazuki Tawaratsumida ◽  
Erin T. Larragoite ◽  
Elizabeth S. C. P. Williams ◽  
Vicente Planelles ◽  
...  
Keyword(s):  
Test Performance ◽  
Learning Algorithm ◽  
Point Of Care ◽  
Method Comparison ◽  
High Specificity ◽  
Turnaround Time ◽  
Analysis Tool ◽  
Point Of Care Test ◽  
Specificity And Sensitivity ◽  
A Cell

AbstractDiagnostic tests that detect antibodies (AB) against SARS-CoV-2 for evaluation of seroprevalence and guidance of health care measures are important tools for managing the COVID-19 pandemic. Current tests have certain limitations with regard to turnaround time, costs and availability, particularly in point-of-care (POC) settings. We established a hemagglutination-based AB test that is based on bi-specific proteins which contain a dromedary-derived antibody (nanobody) binding red blood cells (RBD) and a SARS-CoV-2-derived antigen, such as the receptor-binding domain of the Spike protein (Spike-RBD). While the nanobody mediates swift binding to RBC, the antigen moiety directs instantaneous, visually apparent hemagglutination in the presence of SARS-CoV-2-specific AB generated in COVID-19 patients or vaccinated individuals. Method comparison studies with assays cleared by emergency use authorization demonstrate high specificity and sensitivity. To further increase objectivity of test interpretation, we developed an image analysis tool based on digital image acquisition (via a cell phone) and a machine learning algorithm based on defined sample-training and -validation datasets. Preliminary data, including a small clinical study, provides proof of principle for test performance in a POC setting. Together, the data support the interpretation that this AB test format, which we refer to as ‘NanoSpot.ai’, is suitable for POC testing, can be manufactured at very low costs and, based on its generic mode of action, can likely be adapted to a variety of other pathogens.

scholarly journals Capture-based enrichment of Theileria parva DNA enables full genome assembly of first buffalo-derived strain and reveals exceptional intra-specific genetic diversity

2020 ◽  
Author(s):  
Nicholas C Palmateer ◽  
Kyle Tretina ◽  
Joshua Orvis ◽  
Olukemi O Ifeonu ◽  
Jonathan Crabtree ◽  
...  
Keyword(s):  
Vaccine Development ◽  
De Novo ◽  
High Specificity ◽  
Theileria Parva ◽  
Nucleotide Polymorphisms ◽  
Specificity And Sensitivity ◽  
Genome Wide ◽  
Dna Capture ◽  
High Level ◽  
Genome Assemblies

AbstractTheileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ∼54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is larger than the genome from the reference, cattle-derived, Muguga strain. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average FST, genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species, with clear implications for vaccine development. The DNA capture approach used provides clear advantages over alternative T. parva DNA enrichment methods used previously and enables in-depth comparative genomics in this apicomplexan parasite.

scholarly journals Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Zhang Tie ◽  
Wang Chunguang ◽  
Wei Xiaoyuan ◽  
Zhao Xinghua ◽  
Zhong Xiuhui
Keyword(s):  
Staphylococcus Aureus ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Reaction Tube ◽  
Specificity And Sensitivity ◽  
Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

The loop-mediated isothermal amplification assay for rapid diagnosis of Babesia canis canis infections in dogs

2013 ◽  
Vol 16 (1) ◽  
pp. 131-133 ◽  
Author(s):  
Ł. Adaszek ◽  
M. Jankowska ◽  
M. Kalinowski ◽  
T. Banach ◽  
D. Wułupek ◽  
...  
Keyword(s):  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Babesia Canis ◽  
Canine Babesiosis ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Amplification Assay ◽  
Analytical Laboratories ◽  
Babesia Canis Canis

Abstract The aim of this study was to use a rapid and easy DNA-based test, the loop-mediated isothermal amplification (LAMP), for diagnosis of Babesia canis canis infections in dogs. 10 DNA samples of 18S RNA-A and 10 DNA samples of 18S RNA-B of B. canis canis were used in the study. LAMP method could successfully detect DNA in all examined samples down to 0.1 pg dilution. Obtained results suggest that this method has high specificity and sensitivity and can be applied in analytical laboratories in diagnosis of canine babesiosis.

scholarly journals Multiplex logic processing isothermal diagnostic assays for an evolving virus

2018 ◽  
Author(s):  
Sanchita Bhadra ◽  
Miguel A. Saldaña ◽  
Hannah Grace Han ◽  
Grant L. Hughes ◽  
Andrew D. Ellington
Keyword(s):  
Point Of Care ◽  
Direct Analysis ◽  
Human Saliva ◽  
Ease Of Use ◽  
Molecular Diagnostic ◽  
One Pot ◽  
Diagnostic Assays ◽  
Specificity And Sensitivity ◽  
Isothermal Assay ◽  
Fail Safe

AbstractWe have developed a generalizable ‘smart molecular diagnostic’ capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our one-pot isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR gate signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our platform by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with 20 virions / µl being directly detected in human saliva within 90 minutes, and crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

scholarly journals A new lateral flow assay to detect sIL-2R during T-cell mediated rejection after kidney transplantation

The Analyst ◽  
2021 ◽  
Author(s):  
Lisa K. Seiler ◽  
Rebecca Jonczyk ◽  
Patrick Lindner ◽  
Ncog Linh Phung ◽  
Christine S. Falk ◽  
...  
Keyword(s):  
Kidney Transplantation ◽  
T Cell ◽  
Point Of Care ◽  
High Specificity ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Point Of Care Test ◽  
Specificity And Sensitivity ◽  
Kidney Rejection ◽  
Acute Kidney Rejection

In this work a novel point of care test to detect sIL-2R during acute kidney rejection with high specificity and sensitivity was developed.

scholarly journals A Novel 7-Single Nucleotide Polymorphism-Based Clonotyping Test Allows Rapid Prediction of Antimicrobial Susceptibility of Extraintestinal Escherichia coli Directly From Urine Specimens

2016 ◽  
Vol 3 (1) ◽  
Author(s):  
Veronika Tchesnokova ◽  
Hovhannes Avagyan ◽  
Mariya Billig ◽  
Sujay Chattopadhyay ◽  
Pavel Aprikian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Susceptibility ◽  
Clinical Diagnostics ◽  
Resolving Power ◽  
Nucleotide Polymorphisms ◽  
Typing Method ◽  
Single Nucleotide ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Urine Specimens

Abstract Background.  Escherichia coli is a highly clonal pathogen. Extraintestinal isolates belong to a limited number of genetically related groups, which often exhibit characteristic antimicrobial resistance profiles. Methods.  We developed a rapid clonotyping method for extraintestinal E coli based on detection of the presence or absence of 7 single nucleotide polymorphisms (SNPs) within 2 genes (fumC and fimH). A reference set of 2559 E coli isolates, primarily of urinary origin, was used to predict the resolving power of the 7-SNP-based typing method, and 582 representative strains from this set were used to evaluate test robustness. Results.  Fifty-four unique SNP combinations (“septatypes”) were identified in the reference strains. These septatypes yielded a clonal group resolution power on par with that of traditional multilocus sequence typing. In 72% of isolates, septatype identity predicted sequence type identity with at least 90% (mean, 97%) accuracy. Most septatypes exhibited highly distinctive antimicrobial susceptibility profiles. The 7-SNP-based test could be performed with high specificity and sensitivity using single or multiplex conventional polymerase chain reaction (PCR) and quantitative PCR. In the latter format, E coli presence and septatype identity were determined directly in urine specimens within 45 minutes with bacterial loads as low as 102 colony-forming units/mL and, at clinically significant bacterial loads, with 100% sensitivity and specificity. Conclusions.  7-SNP-based typing of E coli can be used for both epidemiological studies and clinical diagnostics, which could greatly improve the empirical selection of antimicrobial therapy.

scholarly journals Highly specific SNP detection using 2D graphene electronics and DNA strand displacement

2016 ◽  
Vol 113 (26) ◽  
pp. 7088-7093 ◽  
Author(s):  
Michael T. Hwang ◽  
Preston B. Landon ◽  
Joon Lee ◽  
Duyoung Choi ◽  
Alexander H. Mo ◽  
...  
Keyword(s):  
Personalized Medicine ◽  
High Specificity ◽  
Practical Implementation ◽  
Strand Displacement ◽  
Snp Detection ◽  
Single Nucleotide ◽  
Specificity And Sensitivity ◽  
Dna Strand Displacement ◽  
Wide Range ◽  
Dna Strand

Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine.

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