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Salmonella-contaminated foods, especially poultry-derived foods (eggs, chicken meat), are the major source of salmonellosis. Not only in the European Union (EU), but also in the United States, Japan, and other countries, has salmonellosis been an issue of concern for food safety control agencies. In 2005, EU regulation 1003/2005 set a target for the control and reduction of five target Salmonella entericaserovars—S.Typhimurium, S.Enteritidis, S.Infantis, S.Hadar, and S.Virchow—in breeding flocks. Thus, a simple biochip for the rapid detection of any of these five Salmonellaserovars in poultry products may be required. The objectives of this study were to design S.Virchow-specific primers and to develop a biochip for the simultaneous identification of all or any of these five Salmonellaserovars in poultry and poultry products. Experimentally, we designed novel polymerase chain reaction (PCR) primers for the specific detection of S. Virchow, S.Infantis, and S.Hadar. The specificity of all these primers and two known primer sets for S.Typhimurium and S.Enteritidis was then confirmed under the same PCR conditions using 57 target strains and 112 nontarget Salmonellastrains as well as 103 non-Salmonellastrains. Following multiplex PCR, strains of any of these five Salmonellaserovars could be detected by a chromogenic biochip deployed with DNA probes specific to these five Salmonellaserovars. In comparison with the multiplex PCR methods, the biochip assay could improve the detection limit of each of the Salmonellaserovars from N×103cfu/mL to N×102cfu/mL sample in either the pure culture or the chicken meat samples. With an 8-hour enrichment step, the detection limit could reach up to N×100cfu/mL.

Despite the fast boom of thermally activated delayed fluorescence (TADF) emitters bearing borane-based acceptor, so far, no TADF emitter with a direct B–N linkage between N-donor and boryl acceptor has been reported. The latter should simplify the molecular architecture and hence facilitate the synthetic design and versatility. We report here the preparation and characterization of a new series of N-borylated compounds with functional acridine donor unit; namely: ACBM, PACBM, and SACBM. Spectroscopic studies were performed to explore their photophysical properties that exhibited prominent solvatochromism and thermally activated delayed fluorescence. The time-dependent DFT calculation indicated the involvement of substantial intramolecular charge transfer character for which HOMO and LUMO are spatially separated. For compound SACBM, fabrication of green emitting OLED gave CIE chromaticity of (0.22, 0.59) and maximum external quantum efficiency, luminance efficiency and power efficiency of 19.1%, 60.9 cd/A, and 43.6 lm/W, respectively, demonstrating for the first time the highly efficient OLEDs using N-borylated TADF emitters.

The purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA) real-time quantitative polymerase chain reaction (qPCR) to count the viable cells of Lactobacillus gasseriand Lactobacillus salivariusin probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillusspecies were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseriand L. salivariuswere 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseriand L. salivariuswere 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseriand L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillusspecies was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4–5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 μM to 200 μM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable.

AbstractStaphylococcus aureusis one of the major bacterial species that may cause clinical infection and food-poisoning cases. Strains of this bacterial species may produce a series of superantigens (SAgs) (i.e., staphylococcal enterotoxins [SEs], staphylococcal enterotoxin-like toxins, and toxic shock syndrome toxin). In this study, S. aureusstrains from clinical samples and food-poisoning cases in Taiwan were collected; their SAg profiles, and SmaI digestion patterns determined by pulsed-field gel electrophoresis (PFGE), were then analyzed. Results showed that their SAg gene profiles and SmaI digestion patterns of chromosomal DNA were highly diverse. Although PFGE has been used as a criterion standard for typing of S. aureusstrains, and the SAg profiles have been used in combination with PFGE for typing of S. aureusstrains, we found that strains grouped in these combined patterns could be further discriminated by the random amplified polymorphic DNA (RAPD) method. Thus, the combined use of SAg profiles, PFGE, and RAPD patterns permits high discrimination for typing of S. aureusstrains from not only the clinical samples but also the food-poisoning cases. Such a combined method may be used as a highly accurate approach for epidemiological study and tracing of the contamination origin of staphylococcal infections either in hospitals or food-poisoning cases.

Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfHgene encoding a heat shock protein for the iiPCR detection of Salmonellain chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5′ and 3′ ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonellatested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonellastrains tested, including strains of Enterobacteriaceaeclosely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonellaat as low as 100CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.

A total of 16 Bifidobacteriumspecies were assayed by polymerase chain reaction (PCR) and PCR–denaturing gradient gel electrophoresis (PCR–DGGE) methods targeted on a 770-bp region of the tufgene. Based on this sequence, a genus-specific primer set and 12 primer sets for 12 Bifidobacteriumspecies including those previously reported for six probiotic species were developed. On the other hand, when these 16 Bifidobacteriumspecies were subjected to PCR–DGGE analysis, 13 product migration patterns were obtained. PCR products for strains in pairs of B. adolescentis/B. thermophilum, B. longum/B. magnumand B. lactis/B. gallinarummigrated the same distance on the DGGE gel. Combined with species-specific PCR primers specific to B. adolescentis, B. longumand B. lactis, all of the 16 Bifidobacteriumspecies could be identified. In addition, the subspecies of B. animalis, i.e., B. animalisand B. lactis, could be discriminated. This study indicated that the tufgene is highly useful for the molecular detection of different Bifidobacteriumspecies. Using the PCR and PCR–DGGE methods, 16 Bifidobacteriumspecies, including those from probiotic products and those from other origins, could be rapidly identified.

For the brewing industry, the possibility of beer spoilage by lactic acid bacteria (LAB) or by wild yeasts is always a concern. Contamination by certain LAB, such as Lactobacillus brevis, L. casei, L. plantarum, Pediococcus claussenii, P. damnosus,and P. inopinatus,may result in the deterioration of beer quality. Thus, monitoring of these spoilage organisms during the brewing process is critical to quality control. When concentrations of spoilage organisms in the brewing samples are low and, thus, difficult to detect, collection and concentration of these spoilage organisms prior to the application of diagnostic methods are important. Although the membrane filtration method can be used even if filters of the correct pore sizes are chosen, it is still possible that some LAB cells will go through the membrane filters. Another drawback of membrane filtration is membrane fouling, which restricts the beer volume to be filtered. In this study, polyclonal antibodies against beer-spoilage bacteria were prepared by rabbit immunization. The antibodies were then conjugated to magnetic beads for the capture and concentration of major beer-spoilage LAB. The captured cells were subsequently discriminated to species level by a commercially available oligonucleotide array. The combination of immunomagnetic separation with an oligonucleotide array allowed the simultaneous and sensitive detection of several major beer-spoilage LAB species in the brewing samples within 8 hr. This method is accurate and time-saving and can be used for routine monitoring and control of the spoilage organisms during the beer brewing process.

AbstractStaphylococcusspp., including S. aureus, S. intermedius, S. hyicus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. xylosus,and S. carnosus,are major bacterial species associated with food poisoning, and human and veterinary clinics. Traditional methods for the identification of these staphylococci are time-consuming, laborious, or inaccurate. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and polymerase chain reaction (PCR) primers for the detection of the aforementioned Staphylococcusspecies. These primers were proved to be specific for the detection of their corresponding target strains. Furthermore, by using a consensus primer pair, we were able to co-amplify the intergenic region of groES-groELfor these staphylococci. Followed by a chromogenic macroarray system with the specific probes on the plastic chips, these staphylococci in milk products or clinical samples could be simultaneously detected. When the system was used for the inspection of milk or urine samples containing N×100target cells per milliliter of the sample, all these staphylococcal species could be identified after an 8-h pre-enrichment step. This system also allowed the adequate diagnosis of bacteremia, since N×100target cells per milliliter of the blood samples could be detected after a 12-h pre-enrichment. Compared to the multiplex PCR method, this approach has the additional advantage that it allowed the discrimination of more bacterial strains—even some bacterial strains that may generate PCR products with the same molecular sizes.

AbstractStaphylococcus aureusis one of the major pathogens that can cause staphylococcal infection and food poisoning. There are five major classical types of staphylococcal enterotoxins (SEs): SEA, SEB, SEC, SED, and SEE, as well as new SEs or SE-like superantigens (SAgs), such as SEG to SEU. Since many S. aureusstrains harbor more than one SE gene and identification of SEs involved in food poisoning cases is time consuming, we developed a chromogenic macroarray method that allows convenient and simultaneous detection of classical SE genes and a new SE gene (seg), which is phylogenetically highly related to seband sec. Two sets of degenerated primers labeled with biotin were used to co-amplify all SE genes in S. aureusstrains through the polymerase chain reaction (PCR). Afterwards, these biotin-labeled PCR products were hybridized with SE gene-specific probes spotted on the nitrocellulose membrane. When this macroarray was used to detect enterotoxingenic S. aureusin milk or beef homogenate containing 100–104target cells per milliliter or gram of the sample, all six enterotoxin genes could be identified after a 12-hour enrichment step. This macroarray offers clinical and food inspection laboratories a rapid and economical visual method to detect common enterotoxigenic S. aureusstrains.

PCR primers specific for the detection of Lactobacillus acidophilus, Lactobacillus caseigroup, Lactobacillus delbrueckii, and Bifidobacterium longumwere designed based on the elongation factor Tu gene (tuf). The specificity of these four primer sets were confirmed by PCR with 88 bacterial strains of Lactobacillus, Enterococcus, Bifidobacterium, and other bacterial species. Results indicated that these primer sets generated predicted PCR products of 397, 230, 202, and 161 bp for L. acidophilus, L. delbrueckii, L. caseigroup, and B. longum, respectively. Bacterial species other than the target organisms tested did not generate false-positive results. When these four primer sets were combined for the simultaneous detection of the lactic acid bacteria (LAB) in fermented milk products including yogurt, the LAB species listed on the labels of these products could be identified without the preenrichment step. The identification limit for each LAB strain with this multiplex PCR method was N× 103CFU/ml in milk samples. The results of our multiplex PCR method were confirmed by PCR assay using primers based on the 16S rDNA or the 16S–23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. When this multiplex PCR method was used with the determination of counts of total viable LAB and bifidobacteria, the quality of commercial fermented milk products could be assured.

Staphylococcus aureusmay cause foodborne disease outbreaks and staphylococcal infections and is one of the major causes of mastitis. Rapid and reliable methods for detection of this microorganism in milk and other foods are needed. In this study, we designed a primer set from the sequence of the heat shock protein gene htrA, a gene coding for high-temperature-requirement A (HtrA) protein, and used it for real-time PCR detection of S. aureusisolates: 16 reference strains and 40 strains isolated from food-poisoning cases. All strains tested generated positive results. Bacterial strains other than S. aureus, including strains of other Staphylococcusspecies, did not produce positive results. When this primer set was used for the real-time PCR detection of S. aureusin milk and meat samples without the preenrichment step, samples with target cell numbers greater than 103CFU/ml or CFU/g could be detected, indicating the potential quantitative ability of this real-time PCR assay. With a 10-h preenrichment step, however, a detection limit of 1 CFU/ml or CFU/g could be obtained.

Staphylococcal enterotoxins (SEs) are important causative agents in gastroenteritidis and food poisoning cases. They are serologically grouped into five major classical types, i.e., SEA, SEB, SEC, SED, and SEE. In addition, new SEs, such as SEG through SEM, have recently been identified and characterized. In an attempt to survey the distribution of classical and new SEs in organisms responsible for staphylococcal infections in Taiwan, we developed PCR primers for the genes that define the SEK, SEL, and SEM types. Bacterial strains other than sek, sel, and semStaphylococcus aureus, including strains of other Staphylococcusspecies, did not generate any false-positive results when examined with these primers. The expression potential for the sek, sel, and semtypes were also determined by reverse transcription–PCR. Together with the PCR primers specific for the classical SEs and other new SEs, including SEG, SEH, SEI, and SEJ, we surveyed the SE genes in S. aureusstrains isolated from food poisoning cases. For 147 S. aureusisolates originating from food poisoning cases, 109 (74.1%) were positive for one or more SE genes. Of them, the major classical enterotoxin type was sea(28.6%), followed by seb(20.4%), sec(8.2%), and sed(2.0%). For the new SE types, sei(30.6%) was detected the most often, followed by sek(18.4%), sem(12.9%), and sel(8.2%). Also, 64 (43.5%) of the total bacterial strains had more than one enterotoxin gene.