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 A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

2022 ◽  
Vol 12 ◽  
Author(s):  
Katharina Radakovics ◽  
Claire Battin ◽  
Judith Leitner ◽  
Sabine Geiselhart ◽  
Wolfgang Paster ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Ectopic Expression ◽  
High Sensitivity ◽  
High Specificity ◽  
Reporter System ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Definition Of ◽  
The Individual ◽  
Allergen Extracts

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

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 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.

A Preliminary Evaluation of a Prototype Western Blot Confirmatory Test Kit for Syphilis

1994 ◽  
Vol 5 (6) ◽  
pp. 409-414 ◽  
Author(s):  
H Young ◽  
P J Walker ◽  
D Merry ◽  
A Mifsud
Keyword(s):  
Western Blot ◽  
False Positive ◽  
High Sensitivity ◽  
High Specificity ◽  
Confirmatory Test ◽  
Preliminary Evaluation ◽  
Serological Tests ◽  
Western Blot Test ◽  
Specificity And Sensitivity ◽  
Test Kit

A prototype Western blot kit was evaluated as a confirmatory test for syphilis using 131 sera characterized by other serological tests for syphilis. There were 114 treponemal sera (including 94 cases of early syphilis, 83 of which were untreated) and 17 non-treponemal problem sera (11 gave false positive reactions on screening with the TmpA recombinant antigen enzyme immunoassay (EIA), 3 gave false positive fluorescent treponemal antibody absorbed (FTA-abs) tests, and 3 false positive Captia Syphilis G EIA results). Based on the manufacturer's criteria of reactivity in multiple bands for designating a positive result the Western blot test gave a sensitivity of 99.1% (113/114) and a specificity of 88.2% (15/17) when indeterminate reactions were scored positive and 98.2% (112/114) and 100% (17/17) when indeterminate reactions were scored negative. Sensitivity was high in both treated and untreated infection. Corresponding sensitivities for the TPHA and FTA-abs when equivocal reactions were scored negative were 97.5% (111/114) and 99.1% (113/114). The high sensitivity of the FTA-abs in this study is probably due to the large number of untreated primary infections. Our results with the Western blot, confirm earlier studies using ‘in-house’ test systems and, support a role for a commercial Western blot test in the confirmatory diagnosis of syphilis. Further studies are required to confirm the high specificity and sensitivity of the kit in a larger series including a wider variety of non-treponemal cases as well as patients with untreated and treated infection.

scholarly journals Highly Sensitive and Selective Fluorescent Detection of Gossypol Based on BSA-Stabilized Copper Nanoclusters

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 95 ◽  
Author(s):  
Shuangjiao Xu ◽  
Kehai Zhou ◽  
Dan Fang ◽  
Lei Ma
Keyword(s):  
High Performance ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Hplc Method ◽  
Fluorescent Detection ◽  
Copper Nanoclusters ◽  
Highly Sensitive ◽  
The Us ◽  
Fluorescence Quenching Mechanism ◽  
First Time

In this paper, fluorescent copper nanoclusters (NCs) are used as a novel probe for the sensitive detection of gossypol for the first time. Based on a fluorescence quenching mechanism induced by interactions between bovine serum albumin (BSA) and gossypol, fluorescent BSA-Cu NCs were seen to exhibit a high sensitivity to gossypol in the range of 0.1–100 µM. The detection limit for gossypol is 25 nM at a signal-to-noise ratio of three, which is approximately 35 times lower than the acceptable limit (0.9 µM) defined by the US Food and Drug Administration for cottonseed products. Moreover, the proposed method for gossypol displays excellent selectivity over many common interfering species. We also demonstrate the application of the present method to the measurement of several real samples with satisfactory recoveries, and the results agree well with those obtained using the high-performance liquid chromatography (HPLC) method. The method based on Cu NCs offers the followings advantages: simplicity of design, facile preparation of nanomaterials, and low experimental cost.

scholarly journals DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

2015 ◽  
Vol 8 ◽  
pp. MBI.S29736 ◽  
Author(s):  
Kenjiro Nagamine ◽  
Guo-Chiuan Hung ◽  
Bingjie Li ◽  
Shyh-Ching Lo
Keyword(s):  
Safety Concern ◽  
Rapid Development ◽  
High Sensitivity ◽  
High Specificity ◽  
Clostridium Tetani ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Pcr Assays ◽  
Primer Sets ◽  
Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

scholarly journals An alternative PCR assay with high sensitivity and specificity for the detection of swine toxoplasmosis based on the GRA14 gene

2021 ◽  
Author(s):  
Xi He ◽  
Derong Zhou ◽  
Yanwu Sun ◽  
Yuan Zhang ◽  
Xiaogang Zhang ◽  
...  
Keyword(s):  
Detection Method ◽  
Southern China ◽  
Acute Infection ◽  
High Sensitivity ◽  
High Specificity ◽  
Pcr Detection ◽  
Pcr Assay ◽  
Specific Primers ◽  
Specificity And Sensitivity ◽  
Pcr Method

Abstract Background Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect all warm-blooded animals. Infected swine are considered one of the most important sources of T. gondii infection in humans. Rapidly and effectively diagnosing T. gondii infection in swine is essential. PCR-based diagnostic tests have been fully developed, and very sensitive and specific PCR is crucial for the diagnosis of swine toxoplasmosis. Methods To established a high specificity and sensitivity PCR detection method for swine toxoplasmosis, we used T. gondii GRA14 gene as target to design specific primers and established a PCR detection method for swine toxoplasmosis. A total of 5462 blood specimens collected from pigs in 5 provinces and autonomous regions in southern China during 2016–2017 were assessed by the newly established GRA14 gene PCR method. Result Altogether, we used T. gondii GRA14 gene as target to design specific primers and established a high specificity and sensitivity PCR detection method for swine toxoplasmosis; in particular, this PCR method could detect T. gondii tachyzoite DNA in the acute infection phase. The GRA14 gene PCR assay detected a minimum of 2.35 tachyzoites of T. gondii, and it could be used for T. gondii detection in blood, tissue, semen, urine and waste feed specimens. The overall T. gondii infection rate was 18.9% (1033/5462) by the newly established GRA14 gene PCR method. According to statistical analysis among different regions, the positive rates of swine toxoplasmosis in the Shaanxi, Fujian and Guangdong areas in China from 2016 to 2017 were the highest, at 31.7% (44/139), 21.9% (86/391) and 18.8% (874/4645), respectively (χ2 = 84.2, P < 0.0001). Specimens collected in 2017 had a higher positive rate (19.1% or 886/4639) than those collected in 2016 (16.1% or 155/963) (χ2 = 4.5, P < 0.05). Specimens collected in autumn (39.4% or 187/474), spring (22.8% or 670/2940) and winter (18.2% or 129/709) also had higher positive rates than those collected in summer (3.8% or 57/1479) (χ2 = 427.7, P < 0.0001). Conclusions These results indicate that the new PCR method based on the T. gondii GRA14 gene would be useful for the diagnosis of swine toxoplasmosis and that it would facilitate the diagnosis of toxoplasmosis in clinical laboratories.

scholarly journals INDIRECT ELISA FOR DETECTION OF ANTIBODIES TO FMDV NON-STRUCTURAL PROTEINS IN PORCINE SERA

2018 ◽  
pp. 13-20
Author(s):  
A. S. Yakovleva ◽  
A. V. Kanshina ◽  
A. V. Scherbakov
Keyword(s):  
Indirect Elisa ◽  
High Sensitivity ◽  
High Specificity ◽  
Structural Proteins ◽  
Post Inoculation ◽  
Nonstructural Proteins ◽  
Diagnostic Specificity ◽  
Fmd Virus ◽  
Specificity And Sensitivity ◽  
Sensitivity Specificity

An indirect variant of ELISA used for detection of antibodies to nonstructural proteins of the FMD virus in porcine blood sera was developed. The results of the validation showed that the developed method is characterized by high sensitivity, specificity and reproducibility. When testing the blood serum panel obtained from experimentally infected animals, the method allowed to detect antibodies to FMD virus in 7 of 18 sera collected on day 6 post inoculation, in 13 of 19 sera – on day 7 post inoculation, in 16 of 19 sera – on day 8 post inoculation and in all 76 sera obtained on days 9–12 post inoculation. The diagnostic specificity of 3AB-ELISA was 100% when testing 100 knowingly negative blood sera from pigs imported to Russia from Norway. High specificity and sensitivity of the method, established during the development of the method, are confirmed in the course of routine diagnostic tests.

scholarly journals Heterologous immunoassay strategy for enhancing detection sensitivity of banned dye rhodamine B in fraudulent food

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jin Wang ◽  
Xing Shen ◽  
Peng Zhong ◽  
Zhaodong Li ◽  
Qiushi Tang ◽  
...  
Keyword(s):  
Rhodamine B ◽  
High Performance ◽  
High Specificity ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Exposure Level ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Chinese Prickly Ash

Abstract Background The high quality of antibody (Ab) is critical for an immunoassay; usually, an Ab with low affinity is often regarded as a “bad” one in the immunoassay development. How to use a “bad” Ab to develop a highly sensitive immunoassay is still a huge challenge. Methods In this study, a heterologous immunoassay strategy was designed to enhance the sensitivity for the detection of banned dye, rhodamine B (RB), in fraudulent food. The RB Ab could not recognize RB by pairing with homologous coating antigen (Ag). However, the Ab showed unexpected high specificity and sensitivity recognition after being replaced by heterologous coating Ag. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed based on the heterologous strategy. Results The detection limit of icELISA for chilli powder, Chinese prickly ash, hot-pot seasoning, and chilli sauce was 0.002 μg/kg, and the recoveries of the four samples ranged from 76.0 to 102.0%, with the coefficient of variation between 3.9 and 18.8%. Parallel experiment for 20 market samples with high-performance liquid chromatography (HPLC) was performed on to confirm the performance of the practical application of the developed icELISA, and the results of the two methods had good correlation. Molecular modeling inferred that the carboxyl group of hapten and its exposure level played an important role in the hapten-Ab recognition. Conclusions The proposed icELISA can be used for the surveillance screening of RB in a range of seasoning foods, and the heterologous strategy is an effective approach to enhance the sensitivity in an immunoassay.

scholarly journals Title: LAMP Assays for the Simple and Rapid Detection of Clinically Important Urinary Pathogens Including the Detection of Resistance to 3rd Generation Cephalosporins

2021 ◽  
Author(s):  
Lalainasoa Odile RIVOARILALA ◽  
Victor JEANNODA ◽  
Tania CRUCITTI ◽  
Jean Marc COLLARD
Keyword(s):  
Simple Procedure ◽  
High Sensitivity ◽  
High Specificity ◽  
Urine Samples ◽  
Accurate Identification ◽  
Specificity And Sensitivity ◽  
Genes Encoding ◽  
Thermal Cycler ◽  
Tract Infections ◽  
Group 1

Abstract Background: Timely and accurate identification of uropathogens and determination of their antimicrobial susceptibility is paramount to the management of urinary tract infections (UTIs). The main objective of this study was to develop an assay using LAMP (Loop mediated isothermal amplification) technology for simple, rapid and sensitive detection of the most common bacteria responsible for UTIs, as well as for the detection of the most prevalent genes (encoding cefotaximases from CTX-M group 1) responsible for resistance to 3rd generation of cephalosporins. Method: We designed primers targeting Proteus mirabilis, while those targeting Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis and the CTX-M group 1 resistance gene were benchmarked from previous studies. The amplification reaction was carried out in a warm water bath for 60 min at 63±0.5 °C. The amplicons were revealed by staining with Sybr Green I. Specificity and sensitivity were determined using reference DNA extracts spiked in sterile urine samples. The analytical performance of the assays was evaluated directly on pellets of urine samples from patients suspected of UTI and compared with culture.Results: We found a high specificity (100%) for LAMP assays targeting the selected bacteria (P. mirabilis, E. coli, K. pneumoniae, E. faecalis) and the CTX-M group 1 when using DNA extracts spiked in urine samples. The sensitivities of the assays were around 1.5 103 Colony Forming Units (CFU) /mL corresponding to the cut-off value used to define bacteriuria or UTIs in patients with symptoms. Out of 161 urine samples tested, using culture as gold standard, we found a sensitivity of the LAMP techniques ranging from 96 to 100 % and specificity from 95 to 100 %.Conclusion: We showed that the LAMP assays were simple and fast. The tests showed high sensitivity and specificity using a simple procedure for DNA extraction. In addition, the assays could be performed without the need of an expensive device such as a thermal cycler. These LAMP assays could be useful as an alternative or a complementary tool to culture reducing the time to diagnosis and guiding for more effective treatment of UTIs but also as a powerful diagnostic tool in resource-limited countries where culture is not available in primary health care structures.

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