Search

Ultra-fine-ZnO showed low toxicity in complex water matrix containing multiple components such as PBS buffer and the toxic mechanism of ultra-fine-ZnO has not been clearly elucidated. In present study, enhanced antibacterial activity of 200 nm diameter ultra-fine-ZnO in PBS buffer against Bacillus cereus and Escherichia coli were observed in the presence of several organic acids in comparison with ultra-fine-ZnO in PBS buffer alone. These findings indicated that the toxic effects of the ultra-fine-ZnO was dependent on the concentration of released Zn 2+ which was affected by organic acids. The production of reactive oxygen species (ROS) did not responsible to the toxic mechanism of ultra-fine-ZnO which was tested using the antioxidant N -Acetylcysteine (NAC). Indeed, ultra-fine-ZnO induced bacteria cell membrane leakages and cell morphology damages that eventually led to cell death, which were confirmed using propidium monoazide (PMA) in combination with PCR and scanning electron microscopy (SEM). All data gathered herein suggested that released Zn 2+ played a major role in the microbial toxicity of ultra-fine-ZnO. [ABSTRACT FROM AUTHOR]

Membrane-based lateral flow immunochromatographic strip (LFICS) is widely used in various fields because of its simplicity, rapidity (detection within 10 min), and low cost. However, early designs of membrane-based LFICS for preliminary screening only provide qualitative (“yes/no” signal) or semi-quantitative results without quantitative information. These designs often suffer from low-signal intensity and poor sensitivity and are only capable of single analyte detection, not simultaneous multiple detections. The performance of existing techniques used for detection using LFICS has been considerably improved by incorporating different kinds of nanoparticles (NPs) as reporters. NPs can serve as alternative labels and improve analytical sensitivity or limit of detection of LFICS because of their unique properties, such as optical absorption, fluorescence spectra, and magnetic properties. The controlled manipulation of NPs allows simultaneous or multiple detections by using membrane-based LFICS. In this review, we discuss how colored (e.g., colloidal gold, carbon, and colloidal selenium NPs), luminescent (e.g., quantum dots, up-converting phosphor NPs, and dye-doped NPs), and magnetic NPs are integrated into membrane-based LFICS for the detection of target analytes. Gold NPs are also featured because of their wide applications. Different types and unique properties of NPs are briefly explained. This review focuses on examples of NP-based LFICS to illustrate novel concepts in various devices with potential applications as screening tools. This review also highlights the superiority of NP-based approaches over existing conventional strategies for clinical analysis, food safety, and environmental monitoring. This paper is concluded by a short section on future research trends regarding NP-based LFICS. [ABSTRACT FROM AUTHOR]

Abstract: A highly efficient process using iron oxide magnetic nanoparticles (IO)-based immunomagnetic separation of tumor cells from fresh whole blood has been developed. The process involved polymer coated 30 nm IO that was modified with antibodies (Ab) against human epithelial growth factor receptor 2 (anti-HER2 or anti-HER2/neu) forming IO-Ab. HER2 is a cell membrane protein that is overexpressed in several types of human cancer cells. Using a HER2/neu overexpressing human breast cancer cell line, SK-BR3, as a model cell, the IO-Ab was used to separate 73.6% (with a maximum capture of 84%) of SK-BR3 cells that were spiked in 1 mL of fresh human whole blood. The IO-Ab preferentially bound to SK-BR3 cells over normal cells found in blood due to the high level of HER2/neu receptor on the cancer cells unlike the normal cell surfaces. The results showed that the nanosized magnetic nanoparticles exhibited an enrichment factor (cancer cells over normal cells) of 1:10,000,000 in a magnetic field (with gradient of 100 T/m) through the binding of IO-Ab on the cell surface that resulted in the preferential capture of the cancer cells. This research holds promise for efficient separation of circulating cancer cells in fresh whole blood. [Copyright &y& Elsevier]

Salmonella is a foodborne pathogen that has contributed to numerous food safety accidents worldwide, making it necessary to detect contamination at an early stage. A pair of specific primers based on the invA gene of Salmonella was designed for PCR. Target double-stranded DNA (dsDNA) from PCR was purified and denatured at high temperature to obtain target single-stranded DNA (ssDNA). Two carboxyfluorescein-labeled hairpin probes (H1-FAM and H2-FAM) were designed with complementary portions to the ssDNA sequence so that binding could trigger H1-FAM and H2-FAM hybridization chain reaction (HCR) to produce a long dsDNA complex. In this study, graphene oxide (GO) was used in the development of a homogeneous fluorescence detection platform for Salmonella. Using this HCR-GO assay platform, Salmonella detection was completed in 3.5 h. Salmonella was reliably and specifically detected with a limit of detection (LOD) of 4.2 × 101 cfu/mL in pure culture. Moreover, this new HCR-GO assay platform was successfully applied to the detection of Salmonella in artificially contaminated milk with a LOD of 4.2 × 102 cfu/mL. [ABSTRACT FROM AUTHOR]

In this study, a new vancomycin (Van)-modified poly- l -lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d -alanyl- d -alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus , the entFM , cesB , cer , and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 103 cfu/mL, with a dynamic range of detection at 103 to 107 cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 101 cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria. [ABSTRACT FROM AUTHOR]

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers. [ABSTRACT FROM AUTHOR]

A fluorescence assay combined with PCR, catalytic hairpin assembly (CHA), and graphene oxide (GO) was established to detect emetic Bacillus cereus in milk samples. The processes of the assay are not new, but components of the processes make the assay useful. Two partially complementary hairpin probes (H1 and FAM-H2) were designed according to the target single-strand DNA (ssDNA). The CHA reaction could be initiated only by the target ssDNA, which was generated by the denaturation of PCR amplicons. In the absence of the target ssDNA, CHA reaction could not be triggered, which caused the H1 and FAM-H2 adsorbing on the surface of GO and exhibiting a low fluorescence intensity. Addition of the target ssDNA resulted in opening of the hairpin H1 that subsequently hybridized with H2. Then, target ssDNA would be replaced from the H1 and recycled to promote another CHA reaction. Through the CHA reaction, multiple H1-H2 duplexes were generated that could not adsorb on the surface of GO. Thus, a strong fluorescence signal would be obtained. The assay showed a limit of detection for emetic B. cereus of 6.2 × 101 cfu/mL in pure culture and 5.9 × 102 cfu/mL in spiked milk without enrichment. By changing the PCR primer, the assay developed in this study had potential to detect other bacteria. [ABSTRACT FROM AUTHOR]

Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 10² cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 107 cfu/mL. The SD-PMA-mRTPCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products. [ABSTRACT FROM AUTHOR]

Highlights • Wheat germ agglutinin lectin (WGA) was applied as a recognition agent for S. aureus enrichment. • The use of LMS method resulted in a high enrichment efficiency even at low concentrations (100–102 CFU/mL) of S. aureus. • A quantitative and specific detection strategy based on dsDNA stabilized AuNPs was developed for S. aureus detection. • The detection limit of this method was 3.5 × 100 CFU/mL. Abstract A novel 2-step lectin-magnetic separation (LMS) method combined with gold nanoparticle (AuNPs) -based colorimetric system for specifically and quantitatively detecting S. aureus in blood samples was established. In this study, wheat germ agglutinin lectin from Triticum vulgaris (wheat) (WGA) was applied for the first time as a recognition agent in a 2-step magnetic separation method using magnetic nanoparticles (MNPs) for effective and low-cost bacteria enrichment. A quantitative detection strategy based on the long dsDNA amplicons stabilized with AuNPs against salt-induced aggregation was investigated. The 2-step LMS method resulted in a high enrichment efficiency even at low concentrations (100–102 CFU/mL) of S. aureus in pure culture and blood samples. The AuNPs-based colorimetric system was applied for quantitative detection of S. aureus with a limit of detection (LOD) at 3.5 × 100 CFU/mL. The complete process starting with the blood samples treatment, 2-step LMS, and AuNPs-based colorimetric detection took only 7 h. The low cost and sensitive 2-step LMS combined with AuNPs-based colorimetric detection holds considerable potential for early sepsis diagnosis. [ABSTRACT FROM AUTHOR]

Highlights • A fluorescence sensor based on hybridization chain reaction was developed to achieve sensitive detection of Salmonella. • The Fe 3 O 4 nanoparticles were used to isolate the target ssDNA and led to decrease background signal. • The chosen primer and the two hairpin probes gave high selectivity to detect Salmonella. • HCR amplification increased the sensitivity of the method with the detection limit of 7.4 × 101 CFU/mL. Abstract We developed a fluorescence sensor based on hybridization chain reaction (HCR) and Fe 3 O 4 nanoparticles for the detection of Salmonella , common pathogen related to numerous food poisoning outbreaks. In this study, a pair of specific primers based on invA of Salmonella was designed and used in asymmetric polymerase chain reaction (aPCR) to produce a long target single-stranded DNA (ssDNA) that was captured by biotin-probe labeled magnetic beads (MBs) forming the MBs-ssDNA. The target ssDNA triggered HCR amplification with the two carboxyfluorescein (FAM) labeled DNA hairpin reporter probes (H1-FAM, H2-FAM) that enabled the measurements of the fluorescence intensities from the resulting ssDNA∼(H1-FAM ∼H2-FAM) n. The Fe 3 O 4 nanoparticles were used to isolate the target ssDNA which hybridized with the reporter probes and led to decreased background signal that resulted in a detection limit of 7.4 × 101 CFU/mL in buffer and 6.9 × 102 CFU/g in spiked lettuce. The method developed also showed excellent selectivity over several common foodborne pathogens. The Fe 3 O 4 nanoparticles capture coupled with fluorescence detection sensor showed promise for the detection of Salmonella. [ABSTRACT FROM AUTHOR]

A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5 μg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMAqPCR assay was 3.3 × 102 cfu/mL and 4.4 × 102 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6 × 102 cfu/g was obtained in the presence of the Staphylococcus aureus (107 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF. [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]

In this work, a specific, sensitive, and accurate technique was presented for simultaneous detection of Salmonella spp., Shigella spp., and Staphylococcus aureus in food products, three of the more frequent foodborne pathogens that were usually reported in a variety of food matrices. An internal amplification control (IAC) was added in a multiplex PCR (mPCR) reaction system as an indicator of false negative result that can come from the presence of PCR inhibitors in food products. In the presence of inhibitor, no signal would result for the target genes as well as the IAC which results in a positive signal, thereby, eliminating false negative results. To ensure detection of only the viable cells, the effects of sodium deoxycholate (SD) in combination with propidium monoazide (PMA) treatment in the presence of dead cells and viable cells were investigated. Results showed that PMA treatment alone could not effectively inhibit the detection of 10 7 CFU/mL of dead Salmonella Typhimurium , Shigella sonnei , and S. aureus from PCR amplification. However, the SD in combination with PMA treatment gave negative results for PCR amplification of dead S. Typhimurium, S. sonnei, and S. aureus in pure culture and food products. When the developed SD-PMA-mPCR assay in combination with IAC was applied to detect the spiked food (milk, ground beef), the LOD of SD-PMA-mPCR assay for S. Typhimurium , S. sonnei, and S. aureus inoculated individually or inoculated simultaneously into milk or ground beef were 10 1 CFU/mL or 10 1 CFU/g after 15 h enrichment. The results suggested that the SD-PMA-mPCR assay in combination with IAC held promise for the detection of foodborne S. Typhimurium, S. sonnei, and S. aureus. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a spherical, gram-positive, pathogenic bacterium commonly associated with bovine mastitis and clinical infections. It is also recognized as a pathogen that causes outbreaks of food poisoning. The objective of this study was to develop and evaluate a rapid and reliable technique that combines propidium monoazide (PMA) staining with real-time quantitative (q)PCR to detect and quantify viable cells of Staph. aureus in milk powder and meat products. The inclusivity and exclusivity of the assay were evaluated using 58 strains belonging to 14 species. Serial dilutions of Staph. aureus cells were used to establish a standard curve and to confirm the effect of PMA treatment. Milk powder and meat products were used as the spiked foods, and the ability of PMA-qPCR to eliminate nonviable cells was determined in milk powder. Furthermore, meat products were inoculated with different concentrations of Staph. aureus and 105 cfu/g of Bacillus cereus and Salmonella enterica to test the interference by nontarget microorganisms. When PMA treatment was applied before DNA extraction, we were able to eliminate false-positive results with little effect on viable cells. The PMA-qPCR assay was specific and more sensitive than conventional PCR, and the level of detection was 3.0 x 10² cfu/g in spiked milk powder. Additionally, we observed no significant interference for the detection of viable Staph. aureus from other nontarget bacteria. The PMA-qPCR protocol is an effective and rapid method to quantify viable cells of Staph. aureus in food samples. The PMA-qPCR assay is specific and reliable, offering a valuable diagnostic tool for routine analysis in food and clinical diagnostic research at a reasonable cost. [ABSTRACT FROM AUTHOR]

A magnetic solid-phase extraction method (MSPE) was developed to pre-concentrate and cleanup clenbuterol (CLE) from pork muscle. Novel sulfonated polystyrene magnetic nanobeads (spMNBs) were synthesized via a one-pot emulsion copolymerization method by using divinylbenzene, styrene, and sodium styrene sulfonate in the presence of oleic acid-modified and 10-undecylenic acid-modified magnetic ferrofluid. The resulting spMNBs exhibited high adsorption efficiency for CLE and for 10 other common beta-adrenergic agonists, namely, brombuterol, ractopamine, tulobuterol, bambuterol, cimbuterol, mabuterol, clorprenaline, penbutolol, salbutamol, and cimaterol. The adsorption behavior of the spMNBs for CLE was described by the Langmuir equation with a maximum adsorption capacity of 0.41 mg/g. Under the optimized parameters, the extraction of CLE from 0.5 g of pork muscle required 25 mg of the spMNBs at a shortened adsorption time (0.5 min). The proposed MSPE was coupled with colloidal gold nanoparticle-based immunochromatographic assay (MSPE-AuNPIA) for the quantitative detection of CLE residue in pork muscle. The limit of detection and limit of quantification for the pork muscle were 0.10 and 0.24 ng/g, respectively. The intra-day and inter-day assay recoveries at three CLE spiked concentrations ranged from 92.5% to 98.1%, with relative standard deviations ranging from 3.2% to 13.0%. The results of MSPE-AuNPIA were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CLE values obtained with MSPE-AuNPIA agreed with those obtained with LC-MS/MS. [ABSTRACT FROM AUTHOR]

A rapid, reproducible, sensitive and specific combination assay was developed for Escherichia coli O157:H7 comprising sequential immunomagnetic separation (IMS), followed by propidium monoazide (PMA), exclusion by viable cells, internal amplification control (IAC) and polymerase chain reaction (PCR). IMS was used to improve sensitivity, reduce detection time and eliminate false positives. PMA was used to detect viable cells, and IAC was incorporated into the system to eliminate the inhibitors of PCR from the food matrix. In the presence of inhibitors, IAC produced no signal, thereby eliminating false negative results. The results indicated that the IMS cell capture efficiency was about 90% at a concentration of 1 × 106 cfu mL−1 with a detection limit of 2.1 × 102 cfu mL−1 for pure culture. In an unoptimised system, IMS–PMA–PCR with IAC showed a detection limit of 5 × 103 cfu mL−1 in spiked milk over a 240 min assay, faster than conventional methods of detection. [ABSTRACT FROM AUTHOR]

To improve the accuracy of current methods for the detection of Escherichia coli O157:H7 foodborne disease outbreaks, two reagents, propidium monoazide (PMA) and sodium deoxycholate (SD), were utilized to eliminate the interference of dead and injured cells for qPCR that was combined with immunomagnetic separation (IMS) for cell enrichment in an IMS-SD-PMA-qPCR assay. The optimal SD concentration and the optimal incubation time with SD were recorded at 0.1% and 20 min, respectively. The number of bacteria survivors was compared using plate counts, qPCR, PMA-qPCR, and SD-PMA-qPCR assays after the cell suspensions were heat treated at 63 °C or freeze stored at −20 °C. Cell suspensions treated or not treated with PMA and treated with SD resulted in significantly lower number of bacteria survivors than those analyzed without SD treatment which indicated that dead cell DNA was eliminated with SD. More importantly, the number of bacteria survivors from those analyzed with SD-PMA-qPCR showed a direct correlation with those analyzed using the plate counts methods. In addition, in order to improve the limit of detection (LOD) and shorten the detection time, immunomagnetic separation (IMS) was adopted to capture and enrich the target bacteria. Using spiked milk as a matrix, the IMS-SD-PMA-qPCR showed a detection limit of 102 CFU/mL. Significantly, even in the presence of 106 CFU/mL of non-target bacteria, the LOD for the SD-PMA-qPCR with IMS separation for E. coli O157:H7 in spiked milk matrix was recorded at 102 CFU/mL. This combination assay holds promise for the detection of foodborne E. coli O157:H7. [ABSTRACT FROM AUTHOR]

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 102 CFU/mL, as low as 7.5 × 100 CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 101 CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 103 CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples. [ABSTRACT FROM AUTHOR]

A lateral flow strip test was developed for quantitative detection of clenbuterol (CLE) in swine urine in a 10 min total assay time. This new quantitative system for CLE was developed based on the concept of using the ratio of the optical densities (ODs) of the test line (A T) to that of the control line (A C) to offset the inherent heterogeneity of lateral flow test strips (LFTSAT/AC) and the effect of sample matrix. Various parameters such as immunoreaction time, the concentration of analyte, reaction temperature and pH of the reactions, as well as the ionic strength were optimized in urine samples. The assay kinetics based on the immunological reactions indicated achievement of constant signal within 5–10 min of incubation depending upon the CLE concentration. Using the optimized parameters, the linear range for the quantitative detection of CLE using the LFTSAT/AC was between 100 pg/mL and 2500 pg/mL over a 10 min total assay time. The CLE LFTSAT/AC showed a half maximal inhibitory concentration (IC50) of 460 ± 60 pg/mL (n = 5) and a reliable coefficient of correlation (R 2 = 0.996). The CLE test strip exhibited insignificant cross-reaction (Cr) to mabuterol, isoproterenol, salbutamol, terbutaline, brombuterol, ractopamine and bambuterol. The limit of detection (LOD) for swine urine was 220 pg/mL, and recoveries for spiked sample were 102.35% ± 7.66% at 500 pg/mL, 95.07% ± 7.14% at 1000 pg/mL and 100.65% ± 10.21% at 1500 pg/mL, respectively. The intra-and inter-assay precision at 500–1500 pg/mL CLE concentrations showed coefficients of variation (CVs) at <11%. This rapid quantitative LFTSAT/AC system for CLE in spiked urine samples exhibited a coefficient of correlation with traditional ELISA at 0.97 (N = 50). Furthermore, 96% of 90 samples gave a percent recovery of 100 ± 30%. These results indicated that the 10-min colloidal gold-based LFTSAT/AC system is adaptive, sensitive, reproducible, and comparable to traditional ELISA method with the added advantage of being easy to use, inexpensive, and requires non-skilled personnel. The LFTSAT/AC holds promise for field use an on-site detection of CLE in swine urine samples. [ABSTRACT FROM AUTHOR]

Abstract: We report our investigation of the functions of PagN in Salmonella pathogenesis and its potential as a vaccine candidate. Further investigation conducted in this study indicates that the outer membrane protein PagN is important for Salmonella adhesion/invasion of epithelial cells as well as bacterial virulence. When pagN was deleted from Salmonella enterica serovar Typhimurium (S. Typhimurium), the adhesion and invasion of HT-29 epithelial cells was significantly decreased compared with the wild type strain. Mice infected with the pagN mutant strain exhibited less pathological signs in the intestine and survived longer than the wild-type-infected mice. PagN is widely distributed and conserved among clinical isolates of different Salmonella serovars, making PagN a potential vaccine candidate for Salmonella infection. To elucidate the potential of PagN as a vaccine, we expressed and purified recombinant PagN (rPagN). When rPagN was tested in mice, it provided significant protection against Salmonella infection in vivo. In vitro, anti-PagN serum enhanced clearance of Salmonella, indicating a contribution of PagN-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with rPagN. Our preliminary results indicate more functions of PagN in S. Typhimurium virulence as well as its potential as a protective vaccine. [Copyright &y& Elsevier]

Abstract: Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes (Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella. Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 × 106 CFU/ml in pure culture or 1 × 106 CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 102 CFU/ml (4.3 × 102 CFU/ml for S. Typhimurium, 3.7 × 102 CFU/ml for S. Paratyphi B, 7.2 × 102 CFU/ml for S. Typhi) in pure culture and 103 CFU/g (4.3 × 103 CFU/g for S. Typhimurium, 3.7 × 103 CFU/g for S. Paratyphi B, 7.2 × 103 CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S. Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce. [Copyright &y& Elsevier]

The growing use of zinc oxide nanoparticles (ZnO NPs) in various applications has raised many concerns about the potential risks to human health. In this research, the protective effects of cellular oxidative stress inhibitor N-Acetyl-cysteine (NAC) and endoplasmic reticulum (ER) stress inhibitor Salubrinal (Sal) on reproductive toxicity induced by ZnO NPs were investigated. The results showed that application of these two kinds of cell stress inhibitors after oral ingestion of ZnO NPs could prevent the weight loss of pregnant mice; reduce zinc content in the uterus, placenta and fetus; reduce abnormal development of the offspring; and decrease fetal abortion. Furthermore, RT-qPCR, Western blot and immunofluorescence assay results indicated that NAC restored the expression of Gclc , reduced the expression of ATF4 , JNK and Caspase-12 , and decreased the expression of eNOS and IGF-1, in the placenta. Sal decreased the expression of ATF4 , JNK and Caspase-12 , and increased the expression of eNOS and IGF-1 caused by the oral ingestion of ZnO NPs. These results indicated that treatment with NAC and Sal after oral exposure could reduce reproductive and development toxicity caused by ZnO NPs which induced reproductive and development toxicity that was probably caused by the activation of oxide stress and ER stress. • ZnO NPs can cause maternal reproductive toxicity and fetal growth restriction. • ZnO NPs may induced toxicity via endoplasmic reticulum stress and oxide stress. • NAC and Sal can alleviate the reproductive toxicity caused by ZnO NPs. [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]

• LSCM and microplate reader were used to verify the effect of PMA treatment. • Addition of dead bacteria (˜104 CFU/mL) did not affect detection of viable bacteria. • Low concentration of viable bacteria (102 CFU/mL) is detectable in irrigation water. • The method is sensitive and accurate for bacteria detection in irrigation water. Escherichia coli O157:H7 is a kind of waterborne pathogen and recognized as a common pathogenic microorganism in irrigation water. In this study, a quantitative PCR (qPCR) assay with propidium monoazide (PMA) treatment called PMA-qPCR assay was developed to detect the presence of E. coli O157:H7 in natural irrigation water that was more specific, more sensitive, and was unaffected by dead cells compared with traditional and conventional methods. Specific gene, fliC from E. coli O157:H7 was selected to design the unique primer and probe for the assay while propidium monoazide (PMA) was used to effectively prevent the interference of DNA from dead cells. Evaluation of the effect of PMA treatment exhibited permeation of the dead cell membranes that was observed under confocal laser scanning microscope (CLSM). The PMA-qPCR assay efficiently detected viable cells even in the presence of 104 CFU/mL dead cells. Under optimized conditions the limit of detection for viable cells was 102 CFU/mL or 10 CFU/mL in pure culture and spiked irrigation water in a 3 h assay. The PMA-qPCR assay did not show significant difference in the absence of the dead bacteria indicating specificity and selectivity for viable E. coli O157:H7. The PMA-qPCR assay developed in this study has potential for precise and sensitive detection of viable E. coli O157:H7 in irrigation water. [ABSTRACT FROM AUTHOR]