Development of an ELAA kit to detect neomycin in milk

Antibiotics used in livestock production offer various benefits as an antimicrobial agent, growth promoter, and feed effective improvement. However, the abuse of antibiotics leads to antibiotic resistance which may seriously threaten human and animal welfare, and growing levels of antibiotics or antibiotic-resistant bacteria in the environment increase the numbers of drug-resistant infection outbreaks. Therefore, many detection methods have been being developed to quickly assess antibiotic content and its residues in foods. Among many analytical methods, the aptamer-based biosensor has considerable attention for its outstanding advantages such as high specificity, high sensitivity, and good selectivity. We use the ELAA (Enzyme-Linked Aptamer Assay) method - a variant of ELISA - which has a high affinity with neomycin. Firstly, we investigated different buffers to create the Neo-BSA complex. As result, 2-(N-morpholino) ethanesulfonic acid (MES) buffer pH 7 was found with the best results. Next, to help the Neo-BSA complex be fixed well on polystyrene wells, we surveyed various buffers and found the coating buffer (50mM Bicarbonate buffer, pH 9.6) rated as the most suitable for this process. In addition, the quality of the kit is also assessed through competitive ELAA reaction components. Therefore, we have investigated and optimized conditions such as aptamer concentration 25 nM in PBS buffer, and the biotinized aptamers did not need heat treatment prior to joining the reaction. From the results, we have successfully developed a calibration curve for antibiotic residue in milk using the ELAA technique, linear range 0,1 ng/mL and 100 ng/mL. Then, we initially surveyed 20 milk samples found that the ELAA method was consistent with the results from LC-MS/MS was obtained showing no difference between the two methods. We continued to test the samples to determine the kit’s sensitivity and specificity. The results showed that the kit has a specificity and sensitivity of 100%. Finally, LOD and LOQ value had xavg = 0.448; SD = 0.22, LOD = xavg + 3SD = 1.11 (ng / ml); LOQ = x tb + 10SD = 2.65 (ng / mL). We will continue to optimize the kit before being brought to the market.

Download Full-text

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

Nanomaterials ◽

10.3390/nano11051207 ◽

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.

Download Full-text

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

Frontiers in Immunology ◽

10.3389/fimmu.2021.817604 ◽

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.

Download Full-text

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

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/435982 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 9

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.

Download Full-text

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

International Journal of STD & AIDS ◽

10.1177/095646249400500606 ◽

1994 ◽

Vol 5 (6) ◽

pp. 409-414 ◽

Cited By ~ 4

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.

Download Full-text

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

Microbiology Insights ◽

10.4137/mbi.s29736 ◽

2015 ◽

Vol 8 ◽

pp. MBI.S29736 ◽

Cited By ~ 2

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.

Download Full-text

Long Noncoding RNAs as Novel Biomarkers Have a Promising Future in Cancer Diagnostics

Disease Markers ◽

10.1155/2016/9085195 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 96

Author(s):

Ting Shi ◽

Ge Gao ◽

Yingli Cao

Keyword(s):

Noncoding Rnas ◽

High Sensitivity ◽

High Specificity ◽

Body Fluids ◽

Cancer Diagnostics ◽

Long Noncoding Rnas ◽

Detection Methods ◽

Tumor Biomarkers ◽

Biological Functions ◽

Sample Collection

Cancers have a high mortality rate due to lack of suitable specific early diagnosis tumor biomarkers. Emerging evidence is accumulating that lncRNAs (long noncoding RNAs) are involved in tumorigenesis, tumor cells proliferation, invasion, migration, apoptosis, and angiogenesis. Furthermore, extracellular lncRNAs can circulate in body fluids; they can be detected and strongly resist RNases. Many researchers have found that lncRNAs could be good candidates for tumor biomarkers and possessed high specificity, high sensitivity, and noninvasive characteristics. In this review, we summarize the detection methods and possible sources of circulating lncRNAs and outline the biological functions and expression level of the most significant lncRNAs in tissues, cell lines, and body fluids (whole blood, plasma, urine, gastric juice, and saliva) of different kinds of tumors. We evaluate the diagnostic performance of lncRNAs as tumor biomarkers. However, the biological functions and the mechanisms of circulating lncRNAs secretion have not been fully understood. The uniform normalization protocol of sample collection, lncRNAs extraction, endogenous control selection, quality assessment, and quantitative data analysis has not been established. Therefore, we put forward some recommendations that might be investigated in the future if we want to adopt lncRNAs in clinical practice.

Download Full-text

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

10.21203/rs.3.rs-357601/v1 ◽

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.

Download Full-text

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

Veterinary Science Today ◽

10.29326/2304-196x-2018-3-26-13-16 ◽

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.

Download Full-text

Recent advances of fluorescent biosensors based on cyclic signal amplification technology in biomedical detection

Journal of Nanobiotechnology ◽

10.1186/s12951-021-01149-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Hongke Qu ◽

Chunmei Fan ◽

Mingjian Chen ◽

Xiangyan Zhang ◽

Qijia Yan ◽

...

Keyword(s):

Signal Amplification ◽

Low Cost ◽

Rolling Circle Amplification ◽

High Sensitivity ◽

High Specificity ◽

Detection Methods ◽

Research Progress ◽

Rolling Circle ◽

Displacement Reactions ◽

Short Time

AbstractThe cyclic signal amplification technology has been widely applied for the ultrasensitive detection of many important biomolecules, such as nucleic acids, proteins, enzymes, adenosine triphosphate (ATP), metal ions, exosome, etc. Due to their low content in the complex biological samples, traditional detection methods are insufficient to satisfy the requirements for monitoring those biomolecules. Therefore, effective and sensitive biosensors based on cyclic signal amplification technology are of great significance for the quick and simple diagnosis and treatment of diseases. Fluorescent biosensor based on cyclic signal amplification technology has become a research hotspot due to its simple operation, low cost, short time, high sensitivity and high specificity. This paper introduces several cyclic amplification methods, such as rolling circle amplification (RCA), strand displacement reactions (SDR) and enzyme-assisted amplification (EAA), and summarizes the research progress of using this technology in the detection of different biomolecules in recent years, in order to provide help for the research of more efficient and sensitive detection methods. Graphical Abstract

Download Full-text

Detection of alpha-methylacyl-CoA racemase (AMACR), a biomarker of prostate cancer, in patient blood samples using a nanoparticle electrochemical biosensor.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.30_suppl.41 ◽

2012 ◽

Vol 30 (30_suppl) ◽

pp. 41-41

Author(s):

Matthew M. Cooney ◽

Cheryl L. Thompson ◽

Po-Yuan Lin ◽

Kai-Lun Cheng ◽

James D. McGuffin-Cawley ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Patients ◽

High Sensitivity ◽

Intraepithelial Neoplasia ◽

Detection Methods ◽

Assay Method ◽

Vitro Assay ◽

Single Use ◽

Detection And Diagnosis

41 Background: A promising prostate cancer biomarker, alpha-methylacyl-CoA racemase (AMACR), has demonstrated the ability to distinguish cancer from healthy and benign cells with high sensitivity and specificity. However the lack of a good clinical assay has limited its translation into the clinic. Here we report on the development of a single use disposable biosensor for AMACR detection. Methods: This is a very inexpensive, small, single-use disposable sensor that requires only a drop of plasma and connects to a portable device. The biosensor utilizes the reaction of pristanic acid with a substrate that includes AMACR to produce Trans-2,3-dehydropritanayl-CoA plus H2O2. The biosensor utilizes iridium oxide nanoparticle catalyst to oxidize the H2O2 produced in the above metabolic pathway. Thus the oxidation of H2O2 yields a measurable current to quantify AMACR in the sample. This is the first in vitro assay method for AMACR based on this reaction mechanism. Results: In our study including plasma from 9 healthy males, 10 patients with high grade prostatic intraepithelial neoplasia and 5 prostate cancer patients we show 100% accuracy in separating prostate cancer patients from controls as well as those with benign prostate conditions. Conclusions: Our data provides strong evidence for the ability of this biosensor to perform as a reliable assay for prostate cancer detection and diagnosis.

Download Full-text