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Silver metals have long been known to possess antimicrobial properties. Recently, even the nanoparticle version of silver (AgNPs) has also been established as antimicrobials. In this study AgNPs were combined with extracts of the medicinal plant Chinese honeysuckle, Lonicera japonica Thunb. The antimicrobial activity of the AgNPs‐herb was tested against pathogenic Escherichia coli CMCC44113. Using different AgNPs or herb (honeysuckle water extract or HWE) ratios in the presence of a fixed concentration of E. coli CMCC44113, potencies were found to be proportional with concentrations. The antimicrobial activities of AgNPs‐HWE combo were significant enhanced, when compared with solely AgNPs or HWE. Thus, atomic force microscopic and propidium monoazide‐PCR were used to probe the damages caused by AgNPs‐HWE combo on the cell morphology and cell membrane integrity of E. coli. The mechanism of AgNPs‐HWE combo against E. coli may attribute to AgNPs leads to cell wall lysis and damages cell membrane integrity, and thus increases the penetration of HWE into the bacterium, which results in more serious damage to bacterial cells. These findings indicated that AgNPs‐herb was more potent than the AgNPs alone and holds promise for the development of nanoparticle enhanced herbal pharmaceuticals. [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]

Silver metals have long been known to possess antimicrobial properties. Recently, even the nanoparticle version of silver (AgNPs) has also been established as antimicrobials. In this study AgNPs were combined with extracts of the medicinal plant Chinese honeysuckle, Lonicera japonica Thunb. The antimicrobial activity of the AgNPs-herb was tested against pathogenic Escherichia coli CMCC44113. Using different AgNPs or herb (honeysuckle water extract or HWE) ratios in the presence of a fixed concentration of E. coli CMCC44113, potencies were found to be proportional with concentrations. The antimicrobial activities of AgNPs-HWE combo were significant enhanced, when compared with solely AgNPs or HWE. Thus, atomic force microscopic and propidium monoazide-PCR were used to probe the damages caused by AgNPs-HWE combo on the cell morphology and cell membrane integrity of E. coli. The mechanism of AgNPs-HWE combo against E. coli may attribute to AgNPs leads to cell wall lysis and damages cell membrane integrity, and thus increases the penetration of HWE into the bacterium, which results in more serious damage to bacterial cells. These findings indicated that AgNPs-herb was more potent than the AgNPs alone and holds promise for the development of nanoparticle enhanced herbal pharmaceuticals. [ABSTRACT FROM AUTHOR]

An enzyme-linked immunoassay based on dual signal transduction mechanisms has been developed for detection of ovarian cancer biomarker CA125. The immunoassay uses a nanoelectrode array (NEA) chip and absorbance methods for the dual detection. The NEA is used to confirm the optical detection of CA125 that is carried out in a high-binding 96-well plate. An alkaline phosphatase (AP) enzyme was used to label the detection antibody to allow for both the optical and electrochemical detection of CA125. Two kinds of substrates were catalyzed by the AP enzyme. para-Nitrophenylphosphate (PNPP) produces chromogenic para-nitrophenol (PNP), which can be optically detected at 405 nm. para-Aminophenylphosphate (PAPP) produces electroactive para-aminophenol (PAP), which can be detected amperometrically between −0.1 and 0.3 V. The linear ranges have been determined to be 5–1000 U mL−1 and 5–1000 U mL−1 for the optical and electrochemical immunoassays, respectively. The limit of detection of the optical immunoassay is 1.3 U mL−1 and 40 U mL−1 for the optical and electrochemical methods, respectively. [ABSTRACT FROM AUTHOR]

A Ru(phen)32+-doped silica fluorescent nanoparticle (FN)-based immunochromatographic test strip (ICTS) sensor was developed for rapid, high sensitivity, easy to use, and low cost quantitative detection of enrofloxacin (ENR) residues in chicken meat. The fluorescence signal intensity of the FNs at the test line (FIT) and control line (FIC) was determined with a prototype of a portable fluorescent strip reader. Unique properties of Ru(phen)32+ doped silica nanoparticles (e.g., large Stokes shift, high emission quantum yield, and long fluorescence lifetime) were combined with the advantages of ICTS and an easy to make portable fluorescent strip reader. The signal was based on FIT/FIC ratio to effectively eliminate strip to strip variation and matrix effects. Various parameters that influenced the strip were investigated and optimized. Quantitative ENR detection with the FNs ICTS sensor using 80 μL sample took only 20 min, which is faster than the commercial ELISA kit (that took 90 min). The linear range of detection in chicken extract was established at 0.025-3.500 ng/mL with a half maximal inhibitory concentration at 0.22 ± 0.02 ng/mL. Using the optimized parameters, the limit of detection (LOD) for ENR using the FNs ICTS sensor was recorded at 0.02 ng/mL in chicken extract. This corresponds to 0.12 μg/kg chicken meat which is two (2) orders of magnitude better that the maximum residue limits (MRLs) imposed in Japan (10 μg/kg) and three (3) orders of magnitude better than those imposed in China. The intra- and inter-assay coefficient of variations (CVs) were 6.04% and 12.96% at 0.5 ng/mL, 6.92% and 12.61% at 1.0 ng/mL, and 6.66% and 11.88% at 2.0 ng/mL in chicken extract, respectively. The recoveries using the new FNs ICTS sensor from fifty (50) ENR-spiked chicken samples showed a highly significant correlation (R² = 0.9693) with the commercial enzyme-linked immunosorbent assay (ELISA) kit. The new FNs ICTS sensor is a simple, rapid, sensitive, accurate, and inexpensive quantitative detection of ENR residues in chicken meat and extracts. [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]

Picogram levels of antibodies against the protective antigen (PA) of Bacillus anthracis were detected in an automated electrochemical sandwich-type enzyme-linked immunosorbant assay. The antibodies were captured and detected using an 8 × 3 array of 50-μm-diameter cavities. The reagent and sample volumes were as low as 200 nL in a less than 25-min assay from capture to signal generation. The electrochemical detection of the antibodies was demonstrated at 0.05–10 μg/mL containing only 10–5,000 pg antibodies. The limit of detection is 10 fg for a 200-nL sample. Detection of anti-PA immunoglobulin G performed in spiked normal human serum and fresh whole human blood did not show a significant difference from detection in a buffer. The initial automation of the assay involved the use of a digital syringe pump for the delivery of reagents to the capture surface. [ABSTRACT FROM AUTHOR]

The article outlines the benefits of miniaturized amperometric electrochemical systems and their applicability in DNA/RNA assays. Emphasizing recent applications for micro-organisms, the advantages offered by miniatured electrochemical methods over the spectroscopic analyses are discussed. Information on how the diminishing size of detecting electrode improves oligonucleotides detection and molecular recognition techniques is presented.

Abstract: Magneto-hydrodynamics (MHD) driven flow using redox species through a low temperature co-fired ceramic (LTCC) channel with screen printed gold electrodes along its sidewalls has been demonstrated. Aqueous 0.25M K3Fe(CN)6 with 0.25M K4Fe(CN)6 was used to propel 3μL of colored dye through an 18mm long LTCC channel at a maximum flow rate of 0.19mm/s with +0.3V applied across the electrodes in the presence of 0.44T permanent magnet. The channel width was the same as the distance between the two 16–19μm thick gold electrodes at 330μm while the depth was the same as the electrode width at 670μm. Fluid flow through the LTCC channel was controlled with either an electromagnet (∼1.78T) or a permanent magnet (0.44T) such that the current flow running across the gold electrodes was oriented following the right hand rule. No flow out of the opposite reservoir was observed when the current was not oriented perpendicular with the magnetic field. The electrode life span in the LTCC channel using 0.25M K3Fe(CN)6 with 0.25M K4Fe(CN)6 decreased at a rate of 0.14% per hour over 68h of cyclic voltammetry and chronoamperometry. We demonstrated that the LTCC channel flow rate is affected by the nature and concentration of the redox species, solution composition, the applied potential across the electrodes, and the intensity of the magnetic field. Redox MHD driven flow is useful in the development of lab-on-a-chip for micro-total analysis systems. [Copyright &y& Elsevier]

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amine-terminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1 0Pa, which was of two different sequences we call 1 0Pa and 1 0Pa) was used to capture the target (T, which was of two different sequences, T1 sequence 481-590 and T2 sequence 472- 590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1 0Pa, 47 bases were complementary to T1 and T2 at 543-590, while on 1 0Pb, 35 bases were complementary to T1 and T2 at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2 0P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where P1 (P1a and P1b) and P2-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples. [ABSTRACT FROM AUTHOR]

Detecting and enumerating fecal coliforms, especially Escherichia coli, as indicators of fecal contamination, are essential for the quality control of supplied and recreational waters. We have developed a sensitive, inexpensive, and small-volume amperometric detection method for E. coli β-galactosidase by bead-based immunoassay. The technique uses biotin-labeled capture antibodies (Ab) immobilized on paramagnetic microbeads that have been functionalized with streptavidin (bead–Ab). The bead–Ab conjugate captures E. coli from solution. The captured E. coli is incubated in Luria Bertani (LB) broth medium with the added inducer isopropyl β- D-thiogalactopyranoside (IPTG). The induced β-galactosidase converts p-aminophenyl β- D-galactopyranoside (PAPG) into p-aminophenol (PAP), which is measured by amperometry using a gold rotating disc electrode. A good linear correlation ( R2=0.989) was obtained between log cfu mL−1 E. coli and the time necessary to product a specific concentration of PAP. Amperometric detection enabled determination of 2×106 cfu mL−1 E. coli within a 30 min incubation period, and the total analysis time was less than 1 h. It was also possible to determine as few as 20 cfu mL−1 E. coli under optimized conditions within 6–7 h. This process may be easily adapted as an automated portable bioanalytical device for the rapid detection of live E. coli. [ABSTRACT FROM AUTHOR]

Presents an electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of cryptosporidium parvum that is capable of distinguishing dead from live oocysts and suitable for miniaturization and array formats. Capture of synthetic target, the DNA gene for mRNA of heat shock protein 70 for cryptosporidium parvum and cross reactivity tests with targets of other organisms; Characterization of surface at various stages of surface modification on Au macrochips using polarization; Infrared characterization of modified gold macrochips; Prospects for miniaturization, associated detection limits and time of assay.

Develops the mouse Immunoglubolin G model system in determining the self-contained microelectrochemical immunoassay of small volumes. Close proximity of electrodes to modified surfaces; Immunoassay activity of recessed microdisk; Employment of electrochemical desorption in recessed microdisk.

The detection of circulating tumor cells (CTCs) offers a noninvasive method for early-stage cancer diagnosis. Although this is a challenging process, the detection and enumeration of CTCs are essential for assessing cancer progression. In this study, we demonstrated an inexpensive and simple method that used folic acid, FA, as targeting probe to replace antibodies for the enrichment and isolation of ovarian cancer CTCs. Bovine serum albumin (BSA) was decorated on magnetic nanoparticles (MNP) to form BSA-MNP, while FA was conjugated on the BSA-MNP surface through a PEG 2K -linker. The FA-BSA-MNP complex showed efficient capture of ovarian cancer CTCs as low as 20 cells/mL. Moreover, this FA-BSA-MNP complex showed minimal nonspecific adsorption between the modified magnetic nanoprobes and ovarian cancer cells, resulting in high specific enrichment. More importantly, the MNP-BSA-FA complex was nontoxic to ovarian cancer cells. The isolated cancer cells remained highly viable, were readily grown in vitro without significant death rate with a viability of 92.7%, and without microscopically visible contamination. These results suggested that the FA-BSA-MNP complex could successfully be used for the capture of ovarian cancer CTCs from whole blood. Moreover, when combined with immunocytochemical staining method, the FA-BSA-MNP captured CTCs could be differentiated from the blood cells. [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]

Enterococcus faecium YF5, a strain previously isolated from sourdough, was assessed for safety and probiotic potential. Its virulence and antibiotic resistant phenotypes (cytolysin and gelatinase production, antibiotic susceptibility) and genes ( cylA, gelE, ace, agg, esp, and vanA) were surveyed. Results indicated that the tested virulence determinants were nontoxic. In addition, E. faecium YF5 was sensitive to 3 antibiotics such as amoxicillin, vancomycin, and chloramphenicol. Furthermore, results of in vivo animal acute oral toxicity of E. faecium YF5 studies were similar to the control group that indicated no abnormalities. In addition, E. faecium YF5 stably survived in low pH, bile salts, gastric, and intestinal fluids in vitro. Moreover, E. faecium YF5 was found to adhere to human colon cancer cell line HT-29 at 3.39 (±0.67) × 105 CFU/mL. When cocultured with pathogenic organisms ( Enterobacter sakazakii CMCC45402, Escherichia coli CMCC44102, enterohemorrhage Escherichia coli O157: H7 CMCC44828, Salmonella Typhimurium CMCC50071, Shigella flexneri 301, and Shigella sonnei ATCC 29930) and 2 gram-positive strains ( Listeria monocytogenes CMCC54001 and Staphylococcus aureus CMCC 26003), it inhibited these foodborne pathogens with exception of S. aureus. Therefore, E. faecium YF5 can be regarded as a safe strain and it may be used as a probiotic preparation or for microecologics. [ABSTRACT FROM AUTHOR]

A new approach is reported for analysis of ultrasmall volumes. It takes advantage of the versatile positioning of a dispenser to shoot ∼150-pL droplets of liquid onto a specific location of a substrate where analysis is performed rapidly, in a fraction of the time that it takes for the droplet to evaporate. In this report, the site where the liquid is dispensed carries out fast-scan cyclic voltammetry (FSCV), although the detection method does not need to be restricted to electrochemistry. The FSCV is performed at a microcavity having individually addressable gold electrodes, where one serves as working electrode and another as counter/pseudoreference electrode. Five or six droplets of 10 mM [Ru(NH[SUB3])[SUB6]]Cl[SUB3] in 0.1 M KC1 were dispensed and allowed to dry, followed by redissolution of the redox species and electrolyte with one or five droplets of water and immediate FSCV, demonstrating the ability to easily concentrate a sample and the reproducibility of redissolution, respectively. Because this approach does not integrate detection with microfluidics on the same chip, it simplifies fabrication of devices for analysis of ultrasmall volumes. It may be useful for single-step and multistep sample preparation, analyses, and bioassays in microarray formats if dispensing and changing of solutions are automated. However, care must be taken to avoid factors that affect the aim of the dispenser, such as drafts and clogging of the nozzle. [ABSTRACT FROM AUTHOR]

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]

An immunoassay has been developed for the detection of the ovarian cancer biomarker CA-125 by utilizing the chemiluminescence resonance energy transfer to graphene quantum dots. This biosensor shows a wide linear range from 0.1 U mL−1 to 600 U mL−1 with a limit of detection of 0.05 U mL−1 for CA-125 in a buffer solution. [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]

Graphical abstract Highlights • A fluorescence assay has been developed for L. monocytogenes detection by combining aPCR and RCA. • By using universal sequence in aPCR reaction, the assay could be applied for other bacteria detection. • The assay showed an LOD of 4.8 × 101 CFU/mL in pure culture and 4.0 × 102 CFU/g in lettuce. Abstract 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 × 101 CFU/mL in pure culture and 4.0 × 102 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. [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]

Circulating tumour cells (CTCs) draw significant attention as a promising biomarker for cancer prognosis, status monitoring, and metastasis diagnosis. However, the concentration of CTCs in peripheral blood is usually extremely low, thereby requiring enrichment followed by isolation of CTCs prior to detection. An immunomagnetic separation is a promising tool for CTCs enrichment. In this study, a cost‐effective magnetic separation method, based on streptavidin–biotin complexation, was developed and the effects of magnetic beads' size in CTCs capture were compared. Magnetic nanobeads which were 25 nm in diameter lead to highest capture efficiency (82.2%) compared with 150 nm magnetic beads and 1 µm microbeads. Based on the streptavidin–biotin system, 25 nm magnetic nanobeads could capture model CTCs over 80% efficiency even at concentrations as low as ∼25 cells/mL that may represent the actual level of CTCs in peripheral blood of cancer patients. Furthermore, the isolated cells remained robust and healthy showing insignificant changes in morphology and behaviour when cultured for 24 h immediately after capture and isolation. The magnetic nanobeads based on streptavidin–biotin complexation showed promise for the easy and efficient capture and isolation of healthy CTCs for further diagnosis and analysis. [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]

Circulating tumour cells (CTCs) draw significant attention as a promising biomarker for cancer prognosis, status monitoring, and metastasis diagnosis. However, the concentration of CTCs in peripheral blood is usually extremely low, thereby requiring enrichment followed by isolation of CTCs prior to detection. An immunomagnetic separation is a promising tool for CTCs enrichment. In this study, a cost-effective magnetic separation method, based on streptavidin-Cbiotin complexation, was developed and the effects of magnetic beads' size in CTCs capture were compared. Magnetic nanobeads which were 25 nm in diameter lead to highest capture efficiency (82.2%) compared with 150 nm magnetic beads and 1 μm microbeads. Based on the streptavidin-Cbiotin system, 25 nm magnetic nanobeads could capture model CTCs over 80% efficiency even at concentrations as low as ¡«25 cells/mL that may represent the actual level of CTCs in peripheral blood of cancer patients. Furthermore, the isolated cells remained robust and healthy showing insignificant changes in morphology and behaviour when cultured for 24 h immediately after capture and isolation. The magnetic nanobeads based on streptavidin-Cbiotin complexation showed promise for the easy and efficient capture and isolation of healthy CTCs for further diagnosis and analysis. [ABSTRACT FROM AUTHOR]

Various surface modifications of iron oxide magnetic nanoparticles (IOMNs) can improve their stability and long‐term retention time in vivo, expanding applications of biomedical fields. However, whether the long‐term retention of IOMNs coated with different surface modifications has toxic effects remains poorly understood. Here, the toxicity of IOMNs modified with polyethylene glycol (PEG), bovine serum albumin (BSA), and carboxyl group (COOH), forming PEG–IOMNs, BSA–IOMNs, and COOH–IOMNs, respectively, were evaluated in the rats. The high accumulation of PEG–IOMNs and COOH–IOMNs both in the liver and lung, and the high accumulation BSA–IOMNs in blood after 24 day recovery were observed by elemental content analysis. Except individual neutrophils in the local portal area, PEG–IOMNs can also induce cytoplasmic vacuolisation in partial liver cells by histopathological examination. Furthermore, the results of RT‐qPCR showed that PEG–IOMNs, BSA–IOMNs, and COOH–IOMNs can change the transcript levels of most genes related to iron homeostasis, mitochondria apoptosis, inflammatory response, but <2‐fold alteration. COOH–IOMNs seemed to induce normal cell apoptosis more easily than BSA–IOMNs and PEG–IOMNs. In conclusion, BSA–IOMNs had longer‐term retention time in blood. IOMNs coated with PEG and BSA can still induce side effects on the liver. [ABSTRACT FROM AUTHOR]

Various surface modifications of iron oxide magnetic nanoparticles (IOMNs) can improve their stability and long-term retention time in vivo, expanding applications of biomedical fields. However, whether the long-term retention of IOMNs coated with different surface modifications has toxic effects remains poorly understood. Here, the toxicity of IOMNs modified with polyethylene glycol (PEG), bovine serum albumin (BSA), and carboxyl group (COOH), forming PEG-IOMNs, BSA-IOMNs, and COOH-IOMNs, respectively, were evaluated in the rats. The high accumulation of PEG-IOMNs and COOH-IOMNs both in the liver and lung, and the high accumulation BSA-IOMNs in blood after 24 day recovery were observed by elemental content analysis. Except individual neutrophils in the local portal area, PEG-IOMNs can also induce cytoplasmic vacuolisation in partial liver cells by histopathological examination. Furthermore, the results of RT-qPCR showed that PEG-IOMNs, BSA-IOMNs, and COOH-IOMNs can change the transcript levels of most genes related to iron homeostasis, mitochondria apoptosis, inflammatory response, but <2-fold alteration. COOH-IOMNs seemed to induce normal cell apoptosis more easily than BSA- IOMNs and PEG-IOMNs. In conclusion, BSA-IOMNs had longer-term retention time in blood. IOMNs coated with PEG and BSA can still induce side effects on the liver. [ABSTRACT FROM AUTHOR]

Bacillus cereus is a causative agent of an emetic food-borne disease. In this study, visual detection of viable emetic B. cereus was developed using a propidium monoazide (PMA)-asymmetric polymerase chain reaction (asPCR) and unmodified gold nanoparticles (AuNPs). To detect only the viable emetic B. cereus , PMA treatment was selected before DNA extraction to eliminate the false-positive results from dead bacteria. In the presence of viable target bacteria, the long genomic DNA fragments from the cereulide synthetase gene ( cesB ) were produced by asPCR, which could be effectively absorbed onto naked AuNPs via coordination between Au and the nitrogen atoms of the exposed bases. After adding NaCl solution, visual detection with the naked-eye or with UV–vis spectrophotometer was possible within a few minutes. Under the optimum conditions, the limit of detection (LOD) for viable emetic B. cereus reached as low as 9.2 × 10 1 CFU/mL in 0.01 M phosphate-buffered saline and 3.4 × 10 2 CFU/mL in milk, which was adequate to meet the maximum limit imposed by the Commission Regulation (EC) No 2073/2005 (500 CFU/mL). The proposed method also exhibited excellent discrimination against 10 common pathogenic bacteria in milk. The PMA-asPCR-AuNPs colorimetric assay offers a promising application in the detection of low concentrations of food-borne pathogens. [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]

[Display omitted] • A unique Salmonella detection approach that combines PCR and HCR with DLS. • Preliminary exploration of the influence of pH of AuNPs and concentration of SA. • Detection of pathogenic bacteria based on biotin-streptavidin system. Rapid detection of Salmonella in food has always been a research hotspot, which can contaminate milk. In this study, a novel method that combines streptavidin (SA)-modified gold nanoparticles (AuNPs) and dynamic light scattering (DLS) technology for ultrasensitive detection of Salmonella was proposed. First, dual nucleic acid amplification strategies, namely, polymerase chain reaction (PCR) and hybridization chain reaction (HCR), PCR was used to amplify the invA gene from Salmonella to produce the target single-stranded DNA to trigger HCR, whereas HCR was used to produce a long biotin-containing double-strand DNA. After adding the SA-AuNPs probe, the diameter change was measured by DLS. And different characterization methods were also used to verify the feasibility of the established PCR-HCR-DLS strategy. Moreover, under optimal experimental conditions, the developed method showed a limit of detection (LOD) at 100 CFU/mL in both pure culture and spiked skimmed milk. Therefore, the biosensor based on SA-AuNPs and PCR-HCR-DLS showed potential application in detecting Salmonella in milk. [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]

Cronobacter sakazakii , Staphylococcus aureus , and Bacillus cereus are important pathogens contaminating infant food products. In this study, we developed a specific, sensitive, and accurate technique for the simultaneous detection of viable C. sakazakii , S. aureus , and B. cereus in infant food products. An internal amplification control (IAC) was added to the multiplex PCR (mPCR) as indicator of false negative results that are mainly caused by the PCR inhibitors in food products. In addition, to detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the PCR signal from dead cells. The limit of detection (LOD) for the viable cells with or without PMA treatment was 10 4 CFU/mL for C. sakazakii and B. cereus , and 10 2 CFU/mL for S. aureus in pure culture, showing that the PMA treatment did not influence the sensitivity. After 12 h enrichment, the PMA-mPCR-IAC assay could detect as low as 10 1 CFU/g for C. sakazakii and S. aureus , and 10 0 CFU/g for B. cereus in spiked infant food products (infant formula, noodles, milk and rice noodles). This PMA-mPCR-IAC assay we developed hold promise for the simultaneous detection of C. sakazakii , S. aureus , and B. cereus in infant food products. [ABSTRACT FROM AUTHOR]

In this study, the effects of cadmium containing QDs (such as CdSe/ZnS and CdSe QDs) and bulk CdCl 2 in pregnant mice, their fetuses, and the pregnancy outcomes were investigated. It was shown that although the QDs and bulk CdCl 2 were effectively blocked by the placental barrier, the damage on the placenta caused by CdSe QDs still led to fetus malformation, while the mice in CdSe/ZnS QDs treatment group exhibited slightly hampered growth but showed no significant abnormalities. Moreover, the Cd contents in the placenta and the uterus of CdSe QDs and CdSe/ZnS QDs treatment groups showed significantly higher than the CdCl 2 treated group which indicated that the nanoscale size of the QDs allowed relative ease of entry into the gestation tissues. In addition, the CdSe QDs more effectively altered the expression levels of susceptive genes related to cell apoptosis, dysplasia, metal transport, cryptorrhea, and oxidative stress, etc. These findings suggested that the nanoscale size of the QDs were probably more important than the free Cd in inducing toxicity. Furthermore, the results indicated that the outer surface shell coating played a protective role in the adverse effects of QDs on late pregnancy mice. [ABSTRACT FROM AUTHOR]

ZnO nanoparticles (NPs) have been assessed to show adverse effects on the liver, but the molecular mechanisms and the role of nanoparticle properties in these adverse reactions have not been sufficiently studied. In this study, the toxicity of various sizes of ZnO particles (bulk, 90 nm, and 30 nm) that were ingested orally over a period of 3 days were evaluated in mice. The blood biochemistry, hematological analyses, and histopathological evaluation showed that there was apparent toxicity caused by smaller ZnO NPs (30 nm) in liver. The smallest ZnO NPs showed highest accumulation in the mice liver. The RT-qPCR data indicated that 30 nm ZnO NPs can induce significant endoplasmic reticulum (ER) stress responses. The ER stress marker of PERK , eIF2α , ATF4 , Chop , JNK , caspase-12 , caspase-9 , GRP94 , and Bax at the mRNA levels were higher expression in 30 nm ZnO NP than that in bulk or 90 nm ZnO. These findings implied that the smaller ZnO NPs (30 nm) activated ER stress responses that signified severe apoptosis in murine liver. [ABSTRACT FROM AUTHOR]

The goal of this study was to evaluate the viability and gene expression relating to probiotic functionality of encapsulated Lactobacillus plantarum CCTCC M 2014170 during freeze-drying. Sodium alginate with or without inulin was applied as an inner layer and skim milk was used as an outer layer for coating sodium alginate particles. The results indicated that skim milk coated inulin-sodium alginate (ISA) encapsulation beads improved the survival of L. plantarum during exposure to adverse environment. The viability of L. plantarum from ISA encapsulation beads did not change after 2 h incubation in simulant gastric fluid (SGF). In 1% bile salt solution, the L. plantarum from ISA encapsulation beads only had 1.21 log CFU/mL reduction after 2 h treatment. After 7 weeks storage, the survival rate of L . plantarum was 20.89%, which showed reduction from 8.52 to 7.84 log CFU/mL. In addition, all genes relating to probiotic functionality of L. plantarum were similarly but not significantly up-regulated except the plnEF , which exhibited the highest up-regulation at more than 17-fold increase. This study indicated that encapsulation with alginate and skim milk in the presence of inulin before freeze-drying protected and improved the probiotic functionality of L. plantarum from the adverse freeze drying environment. [ABSTRACT FROM AUTHOR]

Sulfo-functionalized iron oxide magnetic nanobeads were employed as an adsorbent for magnetic solid-phase extraction of benzodiazepines in beef. The analytes were determined by high performance liquid chromatography–mass spectrometry. The sulfo-functionalized iron oxide magnetic nanobeads were characterized by transmission electron microscopy and infrared spectroscopy. The optimal pH, ionic strength, volume of sulfo-functionalized iron oxide magnetic nanobeads, and elution solvent were investigated for the isolation procedure. Under the optimized conditions, the limits of detection for diazepam, oxzepam, triazolam, and chlordiazepoxide were 0.79, 0.22, 0.13, and 0.23 ng/kg, respectively. The method was employed for the determination of the analytes in beef. Compared with established methods, magnetic solid phase extraction and high-performance liquid chromatography–tandem mass spectrometry method exhibited high sensitivity, rapid separation, and low cost. [ABSTRACT FROM PUBLISHER]

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]

Salmonella infection could come from eating contaminated meat or raw eggs, and drinking milk or water contaminated by Salmonella enteritidis. Therefore, it is necessary to explore a fast and easy method for the detection of S. enteritidis in these diverse samples. For this purpose, a novel particle size sensing tool was designed for ultrasensitive and accurate S. enteritidis detection. This assay consisted of rolling circle amplification (RCA) with dynamic light scattering (DLS) using gold nanoparticles (AuNPs) modified with DNA probe as DNA-AuNPs as the capture surface into a hybrid RCA-DLS assay combined with asymmetric polymerase chain reaction (aPCR) and subsequent detection. Under optimal experimental conditions, the novel hybrid RCA-DLS assay combined with aPCR for S. enteritidis reached a limit of detection (LOD) as low as 3 × 100 CFU/mL in pure culture. In spiked milk samples, the LOD was 2.0 × 100 CFU/mL without pre-enriched bacteria. The total time of RCA-DLS assay was about 6 h which including genomic DNA extraction, aPCR, RCA and DLS determination. The hybrid RCA-DLS assay combined with aPCR holds promise in the specific and sensitive S. enteritidis detection. [Display omitted] • A universal strategy with using PCR for the amplification of invA gene. • The method used two-step nucleic acid amplification for S. enteritidis detection. • The LOD of this proposed RCA-DLS method was evaluated as low as 10° CFU/mL. [ABSTRACT FROM AUTHOR]

Highlights: [•] We successfully synthesized Au nanstar@SiO2 NPs conjugated with the GOx enzyme. [•] We quantitatively measured glucose level by examining SERS peak of H2O2. [•] SERS sensing assay showed good anti-interference capability against UA and AA. [•] This sensor can be used in the real biological matrix such as saliva. [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]

Pregnant mothers are susceptible to bacterial infections, which may compromise the health of mothers and offspring. Enterococcus faecalis is a ubiquitous species found in food, restaurants, and hospitals where pregnant woman frequently become exposed to this bacterium. However, the survival, distribution, translocation, and corresponding influence of E. faecalis have not been investigated during the pregnancy period, when the mother and fetus are susceptible to bacterial infection. In this study, a fluorescing E. faecalis strain was used to track the fate of the bacterium in pregnant mice. Orally administered E. faecalis were found to survive and disseminate to all regions of the intestinal tract. It also altered the bacterial community structure by significantly decreasing the diversity of Lactobacillus species, impairing the normal structure and function of the intestinal barrier, which may contribute to the bacterial translocation into the blood, spleen, placenta, and fetus. This may affect fetal and placental growth and development. [ABSTRACT FROM AUTHOR]