Fluorescence Detection Method Research Articles

Detection and quantification of C-terminally tagged proteins by in-gel fluorescence

The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.

Analytical Determination of Aflatoxin in Ground Corn Check Samples Completed by Multiple Laboratories over Several Years.

Mycotoxins are toxic molecules produced by multiple fungal species, including Aspergillus and Fusarium. Fungal infection of crops can result in mycotoxins entering the animal and human food supply. Enzyme-linked immunosorbent assays and other immunological assays have been developed to detect mycotoxins in foods. To calibrate the response of those methods, reference materials with known amounts of homogeneously dispersed mycotoxins are often utilized, where the mycotoxin concentrations have been determined using high-performance liquid chromatography coupled with absorbance or fluorescence detection methods, or high-performance liquid chromatography coupled with mass spectrometry detection methods. Therefore, it is important that the analytical methods provide accurate and precise quantitation of mycotoxins. The reference materials must also contain homogeneously dispersed known quantities of mycotoxin. To evaluate the accuracy and precision of mycotoxin reference materials and the analytical methods, quantitative results from multiple laboratories were completed each year for several years on ground corn check samples containing known levels of mycotoxins. Results for the quantitation of aflatoxin-containing corn reference samples are presented in this article.

Development of a CRISPR/Cas12a-based fluorescent detection method of Senecavirus A

BackgroundSenecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread.MethodsIn this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system.ResultsThe method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 106 copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours.ConclusionsImportantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.

Research on Solidification Layer Detection in Coal Transportation Process Based on Improved YOLOv8 Algorithm

Railway transportation is one of the main modes of long-distance coal transportation, and it inevitably causes environmental pollution during the transportation process. In order to improve the environment along the railway and increase the utilization rate of coal resources, this paper proposes a detection algorithm for the scattered coal solidification layer during transportation based on the YOLOv8 model and designs an intelligent recognition model suitable for coal solidification layer detection devices by combining fluorescence detection methods. Through experimental analysis, we found that the model meets the requirements of practical detection and can play a crucial role in environmental protection, with high practical application value.

"On-On-Off" Recyclable Fluorescence Battery for Direct and Selective Detection of Glyphosate and Cu2.

Residues of environmental organophosphorus pesticides (OPs) will seriously endanger human health. Most reported OP sensors utilized the restrictions capacity of OPs on the catalytic capacity of acetylcholinesterase (AChE) to acetylthiocholine chloride (ATCh), which suffers from high costs, weak stability, long reaction time, and unrecyclable. Herein, a recyclable strategy was proposed for selective and sensitive detection of glyphosate (Gly). The weak fluorescence of UIO-66-NH2 at 450 nm was enhanced almost 10-fold after reacting with Gly because of the rotation-restricted emission enhancement mechanism. Moreover, inspired by the process of charging and discharging the batteries, we introduced Cu2+ to chelate with Gly. Because of the strong chelation between Cu2+ and Gly, the Gly was removed from UIO-66-NH2, which resulted in the quenching of fluorescence intensity and making UIO-66-NH2 recycle. This method proposed is fast, recyclable, easily conducted, and with a low 0.33 μM LOD in dd H2O based on 3σ/S. The recovery rates of Gly in tap water ranged from 93.07 to 104.35% within a satisfied 7.75% RSD. The Cu2+ LOD is 0.01 mM based on 3σ/S and 94.37-118.34% recovery rates within 6.48% RSD in tap water. We believe that the findings in this work provide a meaningful and promising strategy to detect Gly and Cu2+ in real samples. This sensor first successfully achieves the recycling use of the material in OP fluorescence detection, which greatly decreases the cost of the designed sensor and reduces the possibility of secondary pollution to the environment, broadens a new circulation dimension of fluorescence detection methods in detecting OPs, and has the potential to remove glyphosate from water. It also provides a method to utilize functionalized metal-organic frameworks to establish various sensors.

Fluorescence sensing probe based on functionalized mesoporous MOFs for non-invasive and detection of dopamine in human fluids

Abnormal amount of dopamine (DA) in human body is closely relate to various diseases, such as Parkinson's disease, pheochromocytoma. Real-time monitoring DA is crucial for disease warning, diagnosis and treatment. Currently, most methods rely on invasive blood testing for detecting DA, which is only completed with the aid of the medical staffs in hospitals. Herein, a non-invasive fluorescence visual strategy is developed for the real-time monitoring DA, based on luminescent nanoparticles and modified mesoporous zeolite imidazole framework (ZIF-8-NH2) dodecahedrons. During the reaction process, DA is enriched through the spatial configuration of ZIF-8-NH2 and hydrogen bonding effect. The luminescence of Cr3+-doped zinc gallate (ZnGa2O4:Cr3+, ZGC) is inhibited by the photo-induced electron transfer (PET) mechanism to realize sensitively detecting DA. The intelligent sensing platform based on the designed fluorescence probe and color recognition system is structured for real-time detection of DA in urine. Furthermore, a skin-fitting hydrogel patch is prepared by combining a fluorescent probe with chitosan, which enables sensitive and accurate detection of DA in sweat without the complex sample pretreatment. The non-invasive fluorescence detection method provides an effective strategy for quantitatively monitoring DA in human fluids.

The Fluorescent Detection of Glucose and Lactic Acid Based on Fluorescent Iron Nanoclusters.

In this paper, a novel fluorescent detection method for glucose and lactic acid was developed based on fluorescent iron nanoclusters (Fe NCs). The Fe NCs prepared using hemin as the main raw material exhibited excellent water solubility, bright red fluorescence, and super sensitive response to hydrogen peroxide (H2O2). This paper demonstrates that Fe NCs exhibit excellent peroxide-like activity, catalyzing H2O2 to produce hydroxyl radicals (•OH) that can quench the red fluorescence of Fe NCs. In this paper, a new type of glucose sensor was established by combining Fe NCs with glucose oxidase (GluOx). With the increase in glucose content, the fluorescence of Fe NCs decreases correspondingly, and the glucose content can be detected in the scope of 0-200 μmol·L-1 (μM). Similarly, the lactic acid sensor can also be established by combining Fe NCs with lactate oxidase (LacOx). With the increase in lactic acid concentration, the fluorescence of Fe NCs decreases correspondingly, and the lactic acid content can be detected in the range of 0-100 μM. Furthermore, Fe NCs were used in the preparation of gel test strip, which can be used to detect H2O2, glucose and lactic acid successfully by the changes of fluorescent intensity.

Fabric phase sorptive extraction increases the accuracy of liquid chromatography-fluorescence detection of zearalenone in food samples

ABSTRACT The accuracy of mycotoxins detection in the food samples has always been controversial and more sensitive methods help better regulatory activities and, therefore, the quality of final products. Zearalenone is a toxic substance from mycotoxins mainly produced by Fusarium fungi that has proven endocrine effects and is potentially carcinogenic. This research aimed to apply the fabric phase sorptive extraction (FPSE) technique to the liquid chromatography- fluorescence detection method to increase the sensitivity and accuracy of zearalenone extraction and determination in food samples. Applying graphene nanoparticles in the sol-gel process modifies the method depending on the amount of nanoparticles, volume and type of the back-extraction solvent, sample pH, ionic strength, and back-extraction time, which are all considered and investigated. At analytical conditions, the limit of quantification (LOQ) 16.57 and detection (LOD) of 4.97 ng/mL were observed. The method’s accuracy was determined by three spiking levels of 100, 150, and 200 ng/mL in the real samples. The mean recovery percentage in wheat were 61.69, 70.56, and 97.95%, and in the rice, 58.13, 66.00, and 102.59, respectively. The average percentages of the relative standard deviation of the instrument for intra-and inter-laboratory repeatability were less than 1.7%. This work provided a superior analytical condition to the conventional detection procedures in food safety laboratories.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

BackgroundInfectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes.ResultsThe study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28.ConclusionsThe established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

Identification of Salmonella Enterica Serovar Typhi DNA by loop-mediated isothermal amplification with fluorescent detection

Introduction. Real-time PCR may be used along with loop isothermal amplification method (LAMP) allowing to conduct the study in 30–40 minutes to diagnose typhoid fever. Several LAMP assay variations for detecting Salmonella enterica serovar Typhi have been described. The studies report that relevant primers were tested on strains specific for Malaysia and China. We attempted to evaluate the LAMP primers described above for identifying S. Typhi strains specific for the Russian Federation and compare their sensitivity and specificity with each other. Materials and methods. A comparative in silico analysis of target sequences was carried out both in the open NCBI database and among the genetic sequences of collection strains at the St. Petersburg Pasteur Institute. Several sets of primers for LAMP amplification of various S. Typhi genome regions were tested. The tested primers amplify the following fragments: SalTyp1 — region of the STY1607 gene, SalTyp2 — region of the STY2879 gene, SalTyp3 — region of the STBHUCCB_38510 gene of the PapD chaperone. Results. In silico analysis of LAMP primers showed that only the SalTyp 3 set has strict specificity for Salmonella enterica serovar Typhi. For the SalTyp 1 and SalTyp 2 an opportunity of false-positive reactions with some E. coli strains was shown. A LAMP method for DNA fluorescent detection of the typhoid fever causative agent was chosen. Assay has been based on a marker gene termed STBHUCCB_38510 and amplification with six specific primers. The detection limit was 20 copies/reaction in reference plasmids and the reaction time lasted for 35 minutes. The specificity of the method was tested on DNA specimens of 20 S. Typhi isolates and 90 strains of other heterologous bacteria from 24 different species. No false positive or false negative results were identified. Conclusion. The developed method can be used in clinical practice for laboratory confirmation of typhoid fever diagnosis as a part of epidemiological monitoring of environmental objects as well as food products.

A fluorescent aptasensor for enzyme-free and sensitive detection of kanamycin based on entropy-driven strand displacement reaction

BackgroundKanamycin is an antibiotic that can easily cause adverse side effects if used improperly. Due to the extremely low concentrations of kanamycin in food, quantitative detection of kanamycin becomes a challenge. As one of the DNA self-assembly strategies, entropy-driven strand displacement reaction (EDSDR) does not require enzymes or hairpins to participate in the reaction, which greatly reduces the instability of detection results. Therefore, it is a very beneficial attempt to construct a highly sensitive and specific fluorescence detection method based on EDSDR that can detect kanamycin easily and quickly while ensuring that the results are effective and stable. ResultsWe created an enzyme-free fluorescent aptamer sensor with high specificity and sensitivity for detecting kanamycin in milk by taking advantage of EDSDR and the high specific binding between the target and its aptamer. The specific binding can result in the release of the promoter chain, which then sets off the pre-planned EDSDR cycle. Fluorescent label modification on DNA combined with the fluorescence quenching-recovery mechanism gives the sensor impressive fluorescence response capabilities. The research results showed that within the concentration range of 0.1 nM–50 nM, there was a good relationship between the fluorescence intensity of the solution and the concentration of kanamycin. Specificity experiments and actual sample detection experiments confirmed that the biosensor could achieve highly sensitive and specific detection of trace amounts of kanamycin in food, with a detection limit of 0.053 nM (S/N = 3). SignificanceTo our knowledge, this is the first strategy to combine EDSDR with fluorescence to detect kanamycin in food. Accurate results can be obtained in as little as 90 min with no enzymes or hairpins involved in the reaction. Furthermore, our enzyme-free biosensing method is straightforward, highly sensitive, and extremely specific. It has many possible applications, including monitoring antibiotic residues and food safety.

Fluorescence emission angular dependence on a nanostructured plasmonic grating

AbstractPlasmonic gratings provide an advantageous platform for fluorescence sensing due to their compatibility with functionalization techniques, imaging detection, and the potential for signal enhancement. Among traditional fluorescence detection methods, microscopes are commonly used tools. The interaction between dye fluorescence processes and the plasmonic modes of the grating strongly depends on the measurement configuration and is influenced by the dispersion and spectral characteristics of the plasmonic modes. In our study, we investigated the angular behavior of the fluorescence emission from ATTO700 dye by varying the collection angle using a standard optical microscope coupled with a spectrometer. Our results show a clear dependence of fluorescence emission in terms of spectral shape on the collection angle that can be attributed to plasmonic mode dispersion.

A Dual-mode platform for the rapid detection of Escherichia coli O157:H7 based on CRISPR/Cas12a and RPA.

Escherichia coli O157:H7 (E. coli O157:H7) is a foodborne pathogenic microorganism that is commonly found in the environment and poses a significant threat to human health, public safety, and economic stability worldwide. Thus, early detection is essential for E. coli O157:H7 control. In recent years, a series of E. coli O157:H7 detection methods have been developed, but the sensitivity and portability of the methods still need improvement. Therefore, in this study, a rapid and efficient testing platform based on theCRISPR/Cas12a cleavage reaction was constructed. Through the integration of recombinant polymerase amplification and lateral flow chromatography, we established a dual-interpretation-mode detection platform based on CRISPR/Cas12a-derived fluorescence and lateral flow chromatography for the detection of E. coli O157:H7. For the fluorescence detection method, the limits of detection (LODs) of genomic DNA and E. coli O157:H7 were 1.8fg/µL and 2.4CFU/mL, respectively, within 40min. Conversely, for the lateral flow detection method, LODs of 1.8fg/µL and 2.4 × 102CFU/mL were achieved for genomic DNA and E. coli O157:H7, respectively, within 45min. This detection strategy offered higher sensitivity and lower equipment requirements than industry standards. In conclusion, the established platform showed excellent specificity and strong universality. Modifying the target gene and its primers can broaden the platform's applicability to detect various other foodborne pathogens.

Fabrication of a ratiometric fluorescent probe based on Tb3+ doped dual-emitting carbon dots for the detection of cytochrome c

Cytochrome c (Cyt-c) was commonly an intrinsic biomarker for a variety of cellular characteristics, such as respiration, energy levels, and apoptosis. Herein, a simple fluorescence sensor was constructed for the detection of Cyt-c in buffer and real serum samples. The carbon dots doped with Tb3+ on the premise of 1-(2-pyridylazo)-2-naphthol (PAN) were fabricated and used as a dual-emission ratiometric fluorescent probe for detecting Cyt-c based on the internal filtering effect (IFE). As a fluorescent probe for ultra-sensitive detection, Cyt-c was quantitatively detected at different concentrations from 1 to 1000 nM. The fluorescent detection method for Cyt-c showed a good linear relationship from 1 to 50 nM, and the limit of detection (LOD) was 0.35 nM. In the recovery range of 101.27–103.39 % in human serum samples, the relative standard deviation (RSD) was less than 3.27 % (n = 3). In the end, the possible structures of CDs were predicted by DFT theoretical simulation calculations. All the results proved the ability of carbon dots as fluorescent probes to detect biomarkers and the application prospects in bioanalysis.

Establishment of the HPLC fluorescence detection method for plasma trace ivermectin and its pharmacokinetics in Bactrian camel.

Ivermectin (IVM), a widely used veterinary anthelmintic, lacks recommended doses for Bactrian camels. This study aims to establish its pharmacokinetics in Bactrian camels, comparing with other livestock. A method for high-performance liquid chromatography fluorescence detection of IVM in plasma was developed. IVM exhibited linear scaling (y=0.6946x+0.0088, R2=0.9988) within 0.025-5ng/mL, with a lower limit of quantification of 25.00pg/mL, high recovery (>70%) and low RSD (<7%). In Bactrian camels, IVM injection showed a low Cmax, extended Tmax and apparent secondary absorption compared to cattle and sheep. Slow absorption and widespread distribution were observed, with peak concentration and area under the curve correlating positively with the dose. This study provides insights into IVM pharmacokinetics in Bactrian camels, informing dose determination and highlighting potential metabolic differences compared to other livestock.

Recent progress in microRNA detection using integrated electric fields and optical detection methods

Low-cost, highly-sensitivity, and minimally invasive tests for the detection and monitoring of life-threatening diseases and disorders can reduce the worldwide disease burden. Despite a number of interdisciplinary research efforts, there are still challenges remaining to be addressed, so clinically significant amounts of relevant biomarkers in body fluids can be detected with low assay cost, high sensitivity, and speed at point-of-care settings. Although the conventional proteomic technologies have shown promise, their ability to detect all levels of disease progression from early to advanced stages is limited to a limited number of diseases. One potential avenue for early diagnosis is microRNA (miRNA). Due to their upstream positions in regulatory cascades, blood-based miRNAs are sensitive biomarkers that are detectable earlier than those targeted by other methods. Therefore, miRNA is a promising diagnostic biomarker for many diseases, including those lacking optimal diagnostic tools. Electric fields have been utilized to develop various biomedical assays including cell separation, molecules detection and analysis. Recently, there has been a great interest in the utility of electric fields with optical detection methods, including fluorescence and surface plasmons toward biomarker detection. This mini review first summarizes the recent development of miRNA as a biomarker. Second, the utility of electric fields and their integration with fluorescence detection methods will be discussed. Next, recent studies that utilized electric fields and optical detection methods will be discussed. Finally, in conclusion, technology gaps and improvements needed to enable low-cost and sensitive biomarker detection in point-of-care settings will be discussed.

Neutron Reflectivity for Testing Graphene Oxide Films Sorption of EuCl3 in Ethanol Solution

Neutron reflectivity (NR) was used to study the sorption of Eu(III) by graphene oxide (GO) films exposed to ethanol solution of EuCl3. Most of the earlier sorption studies have been performed using GO dispersed in solution. In contrast, layered structure of GO films imposes limitations for penetration of ions between individual sheets. The analysis of NR data recorded before and after sorption under vacuum demonstrates an increase of GO film thickness due to sorption by 35–40%. The characterization of chemical state of Eu(III) sorbed by GO films by X‐ray absorption near‐edge structure (XANES) in high‐energy resolution fluorescence detection (HERFD) method at the Eu L3 edge reveals that it remains the same as in anhydrous EuCl3. Analysis of all collected data including reference experiments with bulk GO samples allows to conclude that EuCl3 penetrates into GO interlayers with ethanol solution and remains trapped in interlayers after evaporation of ethanol. Sorption of EuCl3 results in nearly complete amorphization of film and likely formation of voids, thus making NR models based on specific volume of unit cell not valid for quantitative evaluation of Eu sorption. Limitations of NR method must be taken into account in future studies of sorption by thin films.

An off-on fluorescence method for acid phosphatase assay based on the inner filter effect of MnO2 nanosheets on vitamin B2

Fluorescence analysis has attracted much attention due to its rapidity and sensitivity. The present work describes a novel fluorescence detection method for acid phosphatase (ACP) on the basis of inner-filter effect (IFE), where MnO2 nanosheets (MnO2 NSs) and vitamin B2 (VB2) are served as absorbers and fluorophores, respectively. In the absence of ACP, the absorption band of MnO2 NSs overlaps well with the excitation band of VB2, resulting in effective IFE and inhibition of VB2 fluorescence. In the presence of ACP, 2-phospho-L-ascorbic acid trisodium salt (AAP) is hydrolyzed to generate ascorbic acid (AA), which efficiently trigger the reduction of MnO2 NSs into Mn2+ ions, causing the weakening of the MnO2 NSs absorption band and the recovery of VB2 fluorescence. Further investigation indicates that the fluorescence recovery degree of VB2 increases with the increase of ACP concentration. Under selected experimental conditions, the proposed method can achieve sensitive detection of ACP in the ranges of 0.5–4.0 mU/mL and 4.0–15 mU/mL along with a limit of detection (LOD) as low as 0.14 mU/mL. Finally, this method was successfully applied for the detection of ACP in human serum samples with satisfactory recoveries in the range of 95.0 %-108 %.

A fluorescence detection method for postharvest tomato epidermal defects based on improved YOLOv5m.

Tomato quality visual grading is greatly affected by the problems of smooth skin, uneven illumination and invisible defects that are difficult to identify. The realization of intelligent detection of postharvest epidermal defects is conducive to further improving the economic value of postharvest tomatoes. An image acquisition device that utilizes fluorescence technology has been designed to capture a dataset of tomato skin defects, encompassing categories such as rot defects, crack defects and imperceptible defects. The YOLOv5m model was improved by introducing Convolutional Block Attention Module and replacing part of the convolution kernels in the backbone network with Switchable Atrous Convolution. The results of comparison experiments and ablation experiments show that the Precision, Recall and mean Average Precision of the improved YOLOv5m model were 89.93%, 82.33% and 87.57%, which are higher than YOLOv5m, Faster R-CNN and YOLOv7, and the average detection time was reduced by 47.04 ms picture-1. The present study utilizes fluorescence imaging and an improved YOLOv5m model to detect tomato epidermal defects, resulting in better identification of imperceptible defects and detection of multiple categories of defects. This provides strong technical support for intelligent detection and quality grading of tomatoes. © 2024 Society of Chemical Industry.

Establishment of a real‐time fluorescence and visual colorimetric detection method for Staphylococcus aureus based on loop‐mediated isothermal amplification

AbstractA loop‐mediated isothermal amplification (LAMP) method combined with calcein was established for the detection of Staphylococcus aureus. LAMP primers were designed targeting the ASL18_04625 species‐specific gene screened through bioinformatics and PCR analysis. The target gene and detection method demonstrated 100% specificity for S. aureus and no cross‐reactions with other pathogens. For LAMP reactions, the results could be directly observed with the naked eye or evaluated by S‐shaped amplification curves of fluorescence signals. The assay's detection limit was 3.395 × 10−4 ng/μL genomic DNA or 3.21 CFU/mL pure bacterial culture. Furthermore, the target bacteria of 8.5 × 105–8.5 × 100 CFU/mL could be accurately detected in spiked milk samples, and the overall detection process could be completed within 40 min. This method improved the accuracy and convenience for S. aureus detection and provied a dependable tool for the rapid screening of S. aureus.