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Pathogenic bacteria contamination poses a major threat to human health. The detection of low-abundance bacteria in complex samples has always been a knotty problem, and high-sensitivity bacterial detection remains challenging. In this work, a novel magnetic platform with high enrichment efficiency for L. monocytogenes was developed. The magnetic platform was designed by branched polyglutamic acid-mediated indirect coupling of cefepime on magnetic nanoparticles (Cefe-PGA-MNPs), and the specific enrichment of low-abundance L. monocytogenes in real samples was achieved by an external magnet, with a capture efficiency over 90%. A controllable and highly active platinum-palladium nanozyme was synthesized and further introduced in the magnetic nanoplatform for the construction of enzymatic colorimetric biosensor. The total detection time for L. monocytogenes was within 100 min. The colorimetric signals generated by labelled nanozyme were corresponding to different concentrations of L. monocytogenes, with a limit of detection (LOD) of 3.1 × 10 1 CFU/mL, and high reliability and accuracy (with a recovery rate ranging from 96.5% to 116.4%) in the test of real samples. The concept of the developed method is applicable to various fields of biosensing that rely on magnetic separation platforms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A novel sandwich assay for the detection of L. monocytogenes was designed based on antibiotic magnetic separation and enzymatic colorimetry. PEG-mediated cefepime functionalized magnetic nanoparticles (Cefe-PEG-MNPs) was reported for the first time to anchor L. monocytogenes cells with excellent bacterial capture capacity. The capture efficiency of L. monocytogenes in lettuce sample with high concentration (3.1 × 10 6 CFU/mL) was more than 73.8%. Anti-L. monocytogenes monoclonal antibody was adopted as the second anchoring agent to ensure the specificity for L. monocytogenes, which was co-modified with HRP on the surface of gold nanoparticles (AuNPs-HRP/mAb) to form AuNPs-HRP/mAb@L. monocytogenes@Cefe-PEG-MNPs sandwich complexes, and TMB was added to generate a colorimetric signal. The limit of detection in contaminated lettuce, watermelon juice, and fresh meat samples were both 3.1 × 10 2 CFU/mL, and the whole assay takes about 110 min. Based on the above facts, the proposed method has great potential for rapid separation and detection of pathogenic bacteria in food., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Contamination of Listeria monocytogenes (L. monocytogenes) in the environment and food can pose a serious threat to human health, and there is an urgent need to establish sensitive on-situ detection methods to mitigate its hazards. In this study, we have developed a field assay that combines magnetic separation technology with antibody-labeled ZIF-8 encapsulating glucose oxidase (GOD@ZIF-8@Ab) to capture and specifically identify L. monocytogenes while GOD catalyzes glucose catabolism to produce signal changes in glucometers. On the other side, horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) were added to recombined with the H 2 O 2 generated by the catalyst to form a colorimetric reaction system that changes from colorless to blue. The smartphone software was used for RGB analysis to complete the on-site colorimetric detection of L. monocytogenes. This dual-mode biosensor showed good detection performance for the on-site application of L. monocytogenes in lake water and juice samples, both with a limit of detection up to 10 1  CFU/mL and a good linear range of 10 1 -10 6  CFU/mL. Therefore, this dual-mode on-site detection biosensor has a promising application for the early screening of L. monocytogenes in environmental and food samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Listeria monocytogenes (L. monocytogenes), a typical foodborne pathogen, poses a serious threat to public health safety. This stimulates to develop a point-of-care testing (POCT) method to achieve rapid, sensitive detection of L. monocytogenes. In this study, polyethylene glycol (PEG) mediated ampicillin functionalized magnetic beads (Amp-PEG-MBs) was prepared successfully and it achieved high efficiency (>90%) and rapid (5 min) capture for L. monocytogenes at room temperature. The innovative combination of antibody (Ab), glucose oxidase (GOD) and graphene oxide (GO) prepared Ab@GO@GOD for the specific recognition of L. monocytogenes. Finally, Amp-PEG-MBs and Ab@GO@GOD were successfully assembled into Amp-PEG-MBs@L. monocytogenes-Ab@GO@GOD sandwich structure which could catalyze the glucose, and the final detection results were recorded by a blood glucose meter (BGM). Magnetic separation (MS) combined with enzyme-catalyzed sensor (MS-Ab@GO@GOD-BGM) was successfully established to achieve the detection of L. monocytogenes in artificially contaminated juice within 66 min with the limit of detection was 10 1  CFU/mL. This sensor has potential for other pathogens detection by modifying specific antibodies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A new, facile, low-cost, and highly sensitive method for detection of Listeria monocytogenes involving a combination of asymmetric polymerase chain reaction (aPCR) and rolling circle amplification (RCA) had been developed. The aPCR-RCA processes were not new but components of the processes made the assay useful. Twenty-one thymine (21-T) tagged forward primer generated universal twenty-one adenine (21-A) aPCR amplicons after aPCR amplification. A poly-T sequence dumbbell-like RCA template was produced through the blunt-end ligation activity of T4 DNA ligase. After the mixture of aPCR amplicons and dumbbell-like RCA template, the RCA reaction would initiate when the addition of phi29 DNA polymerase, then a large number of G-quadruplex sequences were produced which allowed the intercalation of Thioflavin T (3,6-dimethyl-2-(4-dimethylaminophenyl) benzo-thiazolium cation, THT) for easy fluorescence detection. Under the optimal conditions, the assay showed a limit of detection (LOD) of 4.8 × 10 1  CFU/mL in pure culture and 4.0 × 10 2 CFU/g in spiked lettuce homogenates. By changing the aPCR primer, the aPCR-RCA method developed in this study had a potential to detect other bacteria without the design an RCA template for each bacterium., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Infectious diseases caused by Listeria monocytogenes pose a great threat to public health worldwide. Therefore, a rapid and efficient method for L. monocytogenes detection is needed. In this study, a biotin-exposure-based immunomagnetic separation (IMS) method was developed. That is, biotinylated antibody was first targeted to L. monocytogenes. Then, streptavidin-functionalized magnetic nanoparticles were added and anchored onto L. monocytogenes cells indirectly through the strong noncovalent interaction between streptavidin and biotin. Biotin-exposure-based IMS exhibited an excellent capability to enrich L. monocytogenes. Specifically, more than 90% of L. monocytogenes was captured when the bacterial concentration was lower than 10 4  colony-forming units (CFU)/mL. Importantly, the antibody dosage was reduced by 10 times of that in our previous study, which used antibody direct-conjugated magnetic nanoparticles. Propidium monoazide (PMA) treatment prior to PCR amplification could eliminate the false-positive results from dead bacteria and detected viable L. monocytogenes sensitively and specifically. For viable L.monocytogenes detection, enriched L. monocytogenes was treated with PMA prior to asymmetric PCR amplification. The detection limits of the combined IMS with nucleic acid lateral flow (NALF) biosensor for viable L. monocytogenes detection were 3.5 × 10 3  CFU/mL in phosphate buffer solution and 3.5 × 10 4  CFU/g in lettuce samples. The whole assay process of recognizing viable L. monocytogenes was completed within 6 h. The proposed biotin-exposure-mediated IMS combined with a disposable NALF biosensor platform posed no health risk to the end user, and possessed potential applications in the rapid screening and identification of foodborne pathogens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Immunomagnetic separation (IMS) is an effective tool for the preconcentration and purification of food-borne pathogens from complex food samples because of its high capture efficiency (CE). In conventional IMS, antibodies are usually conjugated on the surface of magnetic beads (MB); the random orientation and conformation changes of antibodies on the MB surface can decrease their bioactivity. Moreover, the Brownian motion of immobilized antibodies is weakened, thereby rendering their binding efficiency with bacteria lower than that of free antibodies. Thus, abundant antibodies are commonly required to ensure high CE for IMS, particularly for large volumes. In this study, a 2-step large-volume magnetic separation (10 mL) was proposed to preconcentrate Listeria monocytogenes from pasteurized milk. First, the biotinylated anti-L. monocytogenes monoclonal antibodies (mAb) were bound with L. monocytogenes in 10 mL of diluted milk through an antigen-antibody interaction, and then streptavidin-labeled MB were used to capture biotin-mAb coated with L. monocytogenes by biotin and streptavidin interaction. Under optimal conditions, the CE of 2-step magnetic separation was >90% with L. monocytogenes concentrations ranging from 8 × 10 0 to 8 × 10 4 cfu/mL, whereas the amount of biotin-mAb was 14 fold lower than that of the conventional IMS method. Coupled with a PCR assay, the detection limit of the proposed method was 8 × 10 0 cfu/mL in pure culture and 8 × 10 1 cfu/mL in pasteurized milk without any pre-enrichment process. Moreover, the overall detection time, including sample preparation, large-volume magnetic separation, and PCR, took less than 7 h. In summary, the developed 2-step large-volume IMS combined with PCR was highly sensitive and low cost and, thus, has considerable potential for the rapid screening of food-borne pathogenic bacteria., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth exhibits ultrahigh sensitivity for detecting disease-related biomarkers using sandwich formats. However, the limit of detection (LOD) of this strategy for Listeria monocytogenes is only around 10(3) CFU/mL, which considerably exceeds the amount of L. monocytogenes commonly present in food products (<100 CFU/g). Herein, we report an improved pELISA method for detection of L. monocytogenes at ultralow concentrations with the sandwich formats using silica nanoparticles carrying poly(acrylic acid) brushes as a "CAT container" to increase enzyme loading for enhancing the detection signal. Under optimal conditions, the proposed pELISA exhibits good specificity and excellent sensitivity for L. monocytogenes with a LOD of 8 × 10(1) CFU/mL in 0.01 M phosphate-buffered saline, via a reaction that can be discriminated by the naked eye. The LOD obtained by this method was 2 and 5 orders of magnitude lower than that of conventional CAT-based pELISA and horseradish peroxidase (HRP)-based conventional ELISA, respectively. Coupled with large-volume immunomagnetic separation, the LOD for L. monocytogenes-spiked lettuce samples reached 8 × 10(1) CFU/g. The improved pELISA also exhibited a great potential in detecting a single cell of L. monocytogenes in 100 μL of solution.

Gold nanoparticle (GNP)-based dynamic light scattering (DLS) assay has been widely used for sensitive detection of small analytes based on analyte binding-induced GNP aggregation. However, the use of this new method to detect large biological objectives, such as pathogenic bacteria, has not been reported. This study is the first to describe a homogeneous GNP-based DLS immunoassay for ultrasensitive detection of Listeria monocytogenes. Compared with small analytes, L. monocytogenes has a larger surface and a higher number of antigen epitopes, which serve as carriers that bind to GNP probes to form "GNP-coated bacteria" complexes. To achieve better analytical performance, various parameters including GNP diameter and concentration, amount of labeled antibodies, and immunoreaction time were systematically investigated and optimized. Under the developed optimum conditions, limit of detection (LOD) for L. monocytogenes reached as low as 3.5×10(1)CFUmL(-1) in 0.01M phosphate-buffered saline. Coupled with a large-volume immunomagnetic separation method, LOD for spiked lettuce samples reached 2.2×10(1)CFUg(-1), which was one order of magnitude lower than the maximum limit imposed in Canada (100CFUg(-1)). The proposed method also exhibited excellent discrimination against 17 common pathogenic bacteria in lettuces. The developed GNP-based DLS immunoassay is highly promising as an approach for detecting large biological objectives., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 10(2) CFU/ml (1.2 × 10(2) CFU/ml for S. Typhimurium, 4.0 × 10(2) CFU/ml for E. coli O157:H7 and 5.4 × 10(2) CFU/ml for L. monocytogenes) in pure culture and 10(3) CFU/g (5.1 × 10(3) CFU/g for S. Typhimurium, 7.5 × 10(3) CFU/g for E. coli O157:H7 and 8.4 × 10(3) CFU/g for L. monocytogenes) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S. Typhimurium, E. coli O157:H7 and L. monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bifidobacterium is well known to have an inhibitory effect on the survival, growth, and proliferation of various foodborne pathogens, but the mechanism of the molecular action of B. longum in blocking the invasion of Listeria monocytogenes is not yet well defined. In the present study, following RNA extraction and cDNA synthesis, differential expression of virulence and stress fitness genes in L. monocytogenes and B. longum was determined by real-time PCR. The results indicate that L. monocytogenes virulence factors, including actA, hly, inlA, and plcA, showed significantly downregulated expression during co-incubation of B. longum and L. monocytogenes in phosphate-buffered saline. The relative mRNA levels of oppA and serpin, two stress fitness genes in B. longum, were significantly higher than for the control group. These results indicate that downregulation of L. monocytogenes virulence factors during co-incubation with B. longum might be responsible for the inhibitory effects.

Featured Application: Rapid magnetic enrichment strategy can significantly improve the sensitivity of multiplex PCR analysis and shorten the pre-concentration time. The simultaneous and sensitive detection of pathogenic Listeria spp. was achieved.Listeria monocytogenes and Listeria ivanovii are important pathogenic Listeria spp. that cause infections in humans and animals. Establishing a rapid and sensitive method for the simultaneous screening of pathogenic Listeria spp. is of great significance for ensuring food safety. Multiplex polymerase chain reaction (mPCR) has been extensively reported to simultaneously detect several pathogens in food with high sensitivity, but a time-consuming pre-enrichment process is necessary. In this study, we report the usage of surface-modified polyethyleneimine-coated positively charged magnetic nanoparticles (PEI-MNPs) for rapid enrichment of pathogenic Listeria spp. through electrostatic interactions. The enrichment process takes only 10 min with high capture efficiency (more than 70%) at a wide pH range and ionic strength. Combined with mPCR analysis, the PEI-MNPs-mPCR strategy can simultaneously, rapidly, and sensitively detect pathogenic Listeria spp. without a time-consuming pre-concentration process. Under the optimal conditions, the detection limits of L. monocytogenes and L. ivanovii in lettuce were both as low as 101 CFU/mL, which was a hundred times lower than that without magnetic enrichment. In conclusion, the magnetic enrichment strategy based on charge interaction combined with mPCR analysis has great application potential in shortening the pre-concentration time of foodborne pathogens and improving the detection sensitivity. [ABSTRACT FROM AUTHOR]

Ultrasensitive and simultaneous quantitative detection of Listeria monocytogenes (L. monocytogenes) that can survive in vacuum packaging and frozen food is critical to public health. Herein, teamed boronate affinity (TBA) magnetically functionalized materials combined with biomass fluorescent probe for L. monocytogenes detection in a sandwich-like strategy was established. Firstly, we prepared new TBA magnetically functionalized materials by reducing the pKa value of phenylboronic acid through the TBA strategy in the form of self-assembled intermolecular B-N coordination bonding. To our knowledge, this is the first study where TBA magnetically functionalized materials were utilized for the identification and capture of bacteria, with a high capture efficiency of over 90.8% in spiked lettuce samples for L. monocytogenes (within 35 min), showing superior capture ability. Then, Anti- L. monocytogenes monoclonal antibody was labeled in biomass-derived fluorescent probe with the help of biotin-streptavidin system to achieve specific and sensitive detection of L. monocytogenes. The sandwich-like strategy shows high sensitivity of designed fluorescence sensing platform for L. monocytogenes with a wide linear range (2.2 × 101–2.2 × 106 CFU/mL), a low limit of detection (2.2 × 101 CFU/mL), and a satisfactory recovery rate (85.19%–106.69%) spiked lettuce sample. Notably, the method was low-cost, general and had strong application prospects in the field of food safety. • The binding pH of APBA was lowered by the TBA strategy. • Provides dual identification of L. monocytogenes with LOD as low as 22 CFU/mL. • Efficient capture and detection in actual food samples is still possible. [ABSTRACT FROM AUTHOR]

In this study, we developed a PAMAM dendrimer-mediated biotin amplified magnetic separation method that was coupled with flow cytometry (FCM) for viable L. monocytogenes detection. PAMAM dendrimer-mediated biotin amplified magnetic separation strategy isolated more than 89.15% ± 1.75%  L. monocytogenes both in PBS solutions and in spiked lettuce samples at bacterial concentration below 10 4 CFU/mL. Propidium monoazide (PMA) treatment prior to PCR amplification eliminated the false-positive results from dead bacteria and detected viable L. monocytogenes sensitively and specifically. In this assay, a pair of specific primers was synthesized for the hly gene of L. monocytogenes that was modified with biotin and FAM (FITC) respectively. After PCR amplification, biotin and FAM (FITC) labeled amplicons were immobilized on the streptavidin coated magnetic microbeads, and the mean fluorescence intensity (MFI) of the microbeads was measured with flow cytometer. Combined PAMAM dendrimer-mediated biotin amplified magnetic separation with FCM assay for viable L. monocytogenes detection, and gave a limit of detection (LOD) as low as 3.5 × 10 1 CFU/mL in PBS and 3.5 × 10 2 CFU/g in spiked lettuce samples. Moreover, the method developed exhibited excellent specificity. Therefore, PAMAM dendrimer-mediated biotin amplified magnetic separation method coupled with FCM assay is a highly promising approach for L. monocytogenes detection. [ABSTRACT FROM AUTHOR]

In this study, vancomycin (Van) functionalized PEGylated-magnetic nanoparticles (Van-PMs) combined with polymerase chain reaction (PCR) showed great potential for highly efficient detection of L. monocytogenes at low infectious dose. Herein, Van-PMs played as universal molecular probes for targeting bacterial cells, while PEG was used to help Van close to D-alanyl- d -alanine (D-Ala- d -Ala) moieties on surface of L. monocytogenes . Under optimum conditions, the developed Van-PMs complexes exhibited extremely high capture efficiency (CE) in a 10 mL bacterium suspension with the value was higher than 90% and 83% in the phosphate-buffered saline (PBS) solution and lettuce samples, respectively. Meanwhile, Gram-negative bacteria slightly influenced CE even under concentrations as high as 10 6 CFU/mL. The assay of Van-PMs-PCR was applied to detect L. monocytogenes with detection limits of 30 CFU/mL and 30 CFU/g in PBS solution and lettuce samples, respectively. The whole experiment was completed within 4 h. This assay demonstrated the potential of this modified method in practical application involving the detection of L. monocytogenes or other Gram-positive pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Salmonella Enteritidis, Eschericha coli O157:H7 and Listeria monocytogenes are important pathogens contaminating fresh vegetables. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in fresh vegetables. To detect viable cells, propidium monoazide (PMA) was applied to eliminate the false-positive results. In addition, an internal amplification control (IAC) was added to the quadruplex PCR as indicator of false negative results that are mainly caused by the PCR inhibitors. The limit of detection (LOD) for the viable cells with or without PMA treatment was 10 2 CFU/mL for S. Enteritidis, 10 3 CFU/mL for E. coli O157:H7 and 10 4 CFU/mL for L. monocytogenes , showing that the PMA treatment did not influence the sensitivity. After 12 h enrichment, the PMA-mPCR-IAC combination assay could detect as low as 10 1 CFU/g for viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in spiked vegetables. The PMA-mPCR-IAC combination assay holds promise for the simultaneous detection of viable S. Enteritidis, E. coli O157:H7 and L. monocytogenes in fresh vegetables. [ABSTRACT FROM AUTHOR]

A challenge for optical fiber biosensor is to achieve ultrahigh sensitivity with narrow full width at half maximum (FWHM) of the spectrum. To address this challenge, an ultrahigh-sensitivity microfiber interferometer fiber ring laser (FRL) biosensor is proposed and investigated for Listeria monocytogenes (L. monocytogenes) detection. The fiber biosensor is composed of a singlemode- tapered no core-singlemode (STNS) fiber configuration, which is functionalized with the anti- L. monocytogenes antibodies. An Erbium Doped Fiber Amplifier is applied to the sensor to excite laser and thus reduce the FWHM of the spectrum, which significantly improved the limit of detection (LoD). The proposed STNS FRL biosensor has excellent reproducibility, specificity and sensitivity for L. monocytogenes. The developed STNS FRL biosensor can directly detect L. monocytogenes cells with LoD as low as 1.0 cell/mL, indicating the capability for detecting single cell of L. monocytogenes. Real lettuce and milk samples have been tested and test result in lettuce and milk samples has deviations within ± 30 % from that of Phosphate-buffered saline (PBS) for L. monocytogenes concentrations vary from 101 to 103 cells/mL(g). The developed STNS FRL biosensor has ultrahigh sensitivity, good stability, reproducibility, and specificity, which has potential applications in diseases/medical diagnostics. • Developed new STNS fiber ring laser configuration capable detecting 1.0 cell/mL Listeria monocytogenes solution. • Provide very narrow FWHM of 0.15 nm due to the introduction of fiber ring laser configuration. • Demonstrated detecting 10 cells/ml Listeria monocytogenes in complex food matrices of real milk and lettuce samples. • The sensor structure has potential for medical diagnostics and food safety inspection. [ABSTRACT FROM AUTHOR]

[Display omitted] • The strategy of Amp-MBs-qPCR was established successfully. • Amp-MBs could identify and enrich bacteria of Listeria spp. with high CE. • The strategy could detect L. monocytogenes with high specificity. • The LOD of L. monocytogenes is 102 CFU/mL in spike milk sample within 2.5 h. Listeria monocytogenes (L. monocytogenes) is a typical food-borne pathogen which poses a serious threat to global public health. Achieving rapid and sensitive detection of pathogens in food is critical to solve food safety problems. In this work, ampicillin functionalized magnetic beads (Amp-MBs) were synthesized and used as a capture probe for detection of L. monocytogenes in milk sample combined with quantitative real-time polymerase chain reaction (qPCR) with high sensitivity and specificity. Amp, as a broad spectrum antibiotic was selected to identify bacteria of the Listeria spp. powerfully, and the hly as a specific gene of L. monocytogenes was selected to establish qPCR for accurate detection. At optimal conditions, the capture efficiency of Amp-MBs for L. monocytogenes (101-106 CFU/mL) and other bacteria of Listeria spp. (105 CFU/mL) was higher than 90 %. The limit of detection of Amp-MBs-qPCR strategy for L. monocytogenes was 101 and 102 CFU/mL in PBS and artificially contaminated milk within 2.5 h, respectively. And this strategy could detect L. monocytogenes from high concentration Listeria spp. with ascendant specificity. These results demonstrated that the Amp-MBs-qPCR strategy was successfully established and applied to the specific detection of L. monocytogenes in milk samples. [ABSTRACT FROM AUTHOR]

Multiplex polymerase chain reaction (mPCR) has been widely used for the simultaneous detection of various target bacteria in vegetables. However, an enrichment period is necessary to improve the sensitivity of the mPCR method. In this paper, large-volume (10 mL) immunomagnetic separation (IMS) combined with mPCR for the rapid detection of Listeria monocytogenes and Listeria ivanovii in lettuce without further enrichment process is reported for the first time. Various parameters that affected the capture efficiency (CE) of IMS, including the amounts of streptavidin and biotinylated anti- Listeria monoclonal antibodies on the surface of magnetic nanobeads, the amount of immunomagnetic beads, immunoreaction time, and magnetic separation time, were systematically investigated. Moreover, the concentrations of primers, PCR conditions, and genomic DNA isolation for mPCR assay were optimized. Under optimum conditions, the CE of large-volume IMS for L . monocytogenes and L . ivanovii was greater than 90% when the concentration of target bacteria was less than 10 6 CFU/mL in pure culture, and was more than 80% when the concentration was below 10 5 CFU/mL in lettuce samples. The limit of detection of IMS combined with mPCR assay reached as low as 1.0 CFU/mL in pure culture and 10 CFU/g in lettuce. The overall assay time, including sample preparation, large-volume IMS, and mPCR assay, took less than 7 h. In summary, the developed large-volume IMS-based mPCR system exhibits great potential for routine screening detection of foodborne pathogenic bacteria for safety monitoring. [ABSTRACT FROM AUTHOR]

Foodborne pathogenic microorganism pose a serious threat to public health security; thus, constructing quick and sensitive methods for early screening foodborne pathogenic microorganism is necessary for human health. Teicoplanin (Teic) is a broad-spectrum biometric molecule, which can recognize Gram-positive (G+) bacteria. In this study, using bovine serum albumin (BSA) and PEG 2k as "carrier" and "linker", respectively. Teic was wired to magnetic beads (MBs) to form the biomimetic dandelion-like magnetic nanoparticles (MBs-PEG-BSA-Teic) to capture Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes). The MBs-PEG-BSA-Teic displayed excellent capture ability of S. aureus (> 93%) and L. monocytogenes (> 86%). After separation, the fluorescence probes were added, forming a "sandwich" structural compounds, namely, MBs-PEG-BSA-Teic~bacteria~fluorescence probes. The fluorescence intensity of the probes was measured by a multimode microplate reader. Consequently, this novel method showed high detection sensitivity to target model bacteria, and the detection limit of S. aureus and L. monocytogenes in PBS buffer solution and juice were achieved 101 CFU/mL, spinach and ground beef samples were achieved 102 CFU/g. The method has easy operation and high sensitivity, and it is less interfered by food matrix or negative bacteria; thus, this method is suitable for early screening G+ pathogen in suspected food samples. • BSA and PEG were used to mediate Teic with MBs, introducing multiple binding sites. • MBs-PEG-BSA-Teic with fluorescence method has low LOD and high specificity. • The method can be used for simultaneous detection of multiple G+ bacteria. [ABSTRACT FROM AUTHOR]

[Display omitted] • The MBs-PEG-BSA-Teic and QBs-PEG-Ab based strategy was successfully established. • The strategy used to detect L. monocytogenes with good sensitivity and specificity. • The dual mediation system can increase the load of Teic and improve sensitivity. Listeria monocytogenes (L. monocytogenes) is a zoonosis pathogen that can cause listeriosis with a high morbidity and mortality. Accordingly, a strategy with high sensitivity and exploitability for detecting L. monocytogenes must be developed. Herein, we introduced a double mediation system based on polyethylene glycol (PEG) and bovine serum albumin (BSA) to prepare teicoplanin (Teic)-functionalized magnetic beads (MBs). This system was then combined with monoclonal antibody-modified quantum dot microspheres (QBs), to develop a rapid and sensitive strategy for detecting L. monocytogenes. Teic, as an antibiotic, mainly bind to Gram-positive bacteria via forming the five-point hydrogen bond between the heptapeptide on its main chain and the terminal D-alanyl-D-alanine (D-Ala-D-Ala) on the cell wall of Gram-positive bacteria. In addition, the antibody guaranteed the specificity for L. monocytogenes detection. The capture efficiency (CE) of MBs-PEG-BSA-Teic for L. monocytogenes at the concentrations ranging from 2.6 × 101 CFU/mL to 2.6 × 104 CFU/mL exceeded 92% in phosphate-buffered saline (PBS) and 89% in the spiked ground beef samples. The limit of detection of the strategy for L. monocytogenes was 2.6 × 101 CFU/mL in both PBS and spiked ground beef samples. Therefore, the proposed strategy is capable of detecting L. monocytogenes with a high specificity and an excellent sensitivity. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen which can cause significant threats to human health, thus, a highly specific and accurate method for detection in food is urgently needed. In this study, an enzyme-linked aptasensor with rolling circle amplification (ELARCA) assay was developed for the sensitive and specific detection of L. monocytogenes. The assay was based on the competition between the aptamer (that was specific for L. monocytogenes) that was bound to the biotin probe 1 (BP1~A) and the RCA probe that was complementary to the BP1. Addition of the bacteria released the BP1 making it available for the RCA probe which initiated the RCA process. In the presence of the RCA buffer, multiple copies of the DNA were formed which attached the biotin probe 3 (BP3). The biotin in BP3 interacted with streptavidin labeled with horse radish peroxidase (SA-HRP) to complete the assay complex. In the presence of the enzyme substrate, HRP produced a chromophore that led to the colorimetric detection. Under the optimal conditions, the ELARCA assay for L. monocytogenes showed a limit of detection (LOD) of 4.6 × 102 CFU/mL in pure culture, which was three orders of magnitude higher than without RCA. The ELARCA assay in spiked fresh lettuce showed an LOD of 6.1 × 103 CFU/g. The method developed was fast, low-cost, sensitive, and highly specific for L. monocytogenes. By changing the specificity of the aptamer, the proposed ELARCA method provided a potential platform for the detection of other pathogenic bacteria. • An ELARCA assay was developed for L. monocytogenes detection. • The LOD for L. monocytogenes was 102 CFU/mL in pure culture and 103 CFU/g in spiked lettuce. • The ELARCA method provided a platform for other pathogenic bacteria. [ABSTRACT FROM AUTHOR]