One-step duplex RT-droplet digital PCR assay for the detection of norovirus GI and GII in lettuce and strawberry.

The study was designed to develop a sensitive one-step duplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) to detect norovirus genogroup I and II (NoV GI and GII) in lettuce and strawberry. The specificity, sensitivity, repeatability and robustness of the assay was compared with RT-qPCR. The lowest concentration detected by RT-ddPCR for NoV GI and NoV GII were 4.68 and 8.47 copies/µL respectively, much lower than that of RT-qPCR with a number of 46.8 and 84.7 copies/µL, respectively. Lettuce and strawberry samples were artificially contaminated with NoV GI and GII suspensions, with inoculum size of 3.00 × 106 to 1.70 × 108 copies and 4.80 × 105 to 2.50 × 107 copies, respectively. Strawberry spiked with low inoculum size revealed positive results by RT-ddPCR, while recorded negative by RT-qPCR. Meanwhile, RT-ddPCR also showed a higher average recovery rate for NoV in lettuce and strawberry than RT-qPCR.The limit of detection (LoDs) of RT-ddPCR for NoVs in lettuce was 2.32 × 104 copies/25g (NoV GI) and 2.36 × 104 ciopies/25g (NoV GII), and that in strawberry was 2.56 × 104 copies/25g (NoV GI) and 2.64 × 104 ciopies/25g (NoV GII), which were 10 folds lower than that of RT-qPCR. The developed duplex RT-ddPCR assay exhibited stability and increased capacity to resist inhibitors in food samples with low concentration of NoV, making it a reliable method to avoid false negative result as opposed to RT-qPCR. In conclusion, one-step RT-ddPCR method developed in this study is pertinent in detecting foodborne virus such as NoV.

The effects of bioactive compounds from blueberry and blackcurrant powders on the inhibitory activities of oat bran pastes against α-amylase and α-glucosidase linked to type 2 diabetes.

The α-amylase and α-glucosidase inhibitory activities by extracts of oat bran, blueberry and blackcurrant powders, as well as oat bran pastes supplemented 25% of blueberry and blackcurrant powder, were studied by measuring their half inhibitory (IC50) concentrations. Addition of blueberry or blackcurrant powder into oat bran paste increased α-amylase and α-glucosidase inhibitory activities with a decrease in IC50 values. The main anthocyanidin content was measured by pH differential method and the potential inhibitory mechanisms of these extracts were also investigated by detailed inhibition kinetics and docking simulations. The results showed that: (1) cyanidin and delphinidin were the main anthocyanidin profiles in extracts; (2) only blackcurrant powder was a competitive inhibitor, while other extracts were all mixed-type inhibitors against α-amylase; (3) both blueberry- and blackcurrant-enriched pastes were competitive inhibitors, while other extracts were all mixed-type inhibitors towards α-glucosidase; (4) the α-amylase and α-glucosidase inhibitory activities by extracts were potentially driven by hydrogen bonding, cyanidin-3-glucoside and delphinidin-3-glucoside had stronger binding affinity compared to malvidin-3-glucoside and cyanidin-3-rutinside. This study suggested supplementary of blueberry and blackcurrant with oat bran might be a potential source of bioactive products for antidiabetic activity.

Low-Cost Method Generating In Situ Anaerobic Conditions on a 96-Well Plate for Microbial Fermentation in Food Research.

Commercial tools and instruments have been developed for a screening study of microbial fermentation, but they are expensive and mostly confined to aerobic fermentation only. There is little development on the generation of anaerobic conditions directly on a 96-well plate. This report proposed a simple and versatile microbial fermentation system known as OVAMO that makes use of Oxyrase, vacuum, and mineral oil to generate an in situ anaerobic environment on a 96-well plate for at least 48 h. The practicality of OVAMO in anaerobic fermentation experiments used for functional food research was validated by a prebiotic screening study of different carbohydrates by Bifidobacterium longum subsp. infantis. The OVAMO system provides a less expensive but effective way to conduct a microbial fermentation screening study that requires anaerobic conditions without the need for atmospheric control by external devices.

The diploid genome of the only sclerotia-forming wild-type species in the genus Pleurotus -Pleurotus tuber-regium - provides insights into the mechanism of its biomass conversion from lignocellulose substrates.

Pleurotus tuber-regium (Fr.) Singer, being a white-rot fungus, is widely used for food and medicine in the Asia-Pacific region. In this study, we sequenced and annotated the genome of a dikaryon P. tuber-regium wild strain to provide a better understanding of the carbohydrate-active enzymes (CAZymes) involved in the bio-conversion of lignocellulose to beta-glucan reserves in this sclerotia-forming Pleurotus mushroom with reference to enzyme participated in cellulosic compound breakdown and glucan reserve biosynthesis. The present genomic data provides new insights for lignocellulose bioconversion of white-rot fungus for the genus Pleurotus.

Development of a recombinase polymerase amplification assay for Vibrio parahaemolyticus detection with an internal amplification control.

A novel RPA-IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 103 CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA-IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

Multiplex TaqMan locked nucleic acid real-time PCR for the differential identification of various meat and meat products.

Meat adulteration incidents have been reported frequently over the past few years, and the corresponding traceability issues attracted much more attention due to the customer's demands and administration's responsibility. Therefore, it is important to develop high-throughput and rapid detection methods to identify the specific sources from meat samples. In this study, a multiplex TaqMan locked nucleic acid real-time polymerase chain reaction assay (MLNA-RT-PCR) was developed to simultaneously detect multiple meat sources (duck, pork, beef and chicken). PCR primers and TaqMan-LNA probes were designed based on species-specific mitochondrial gene sequences, and the MLNA-RT-PCR was developed and optimized for better performance. The specificity of this assay was verified through identifying unrelated (sheep, horse, deer, donkey, rabbit, goose, goat, shrimp, salmon and maize) mitochondrial DNA as species-specific targets. The detection limit for MLNA-RT-PCR reached to the level of 0.01% of each species. The assay was then used to identify the meat sources of commercial meat and meat-derived products that were obtained from markets in Shantou, and the results were 98% consistent with that obtained from detection based on the national standard. In conclusion, this MLNA-RT-PCR is a high-throughput, sensitive and specific method that can be used to identify multiple meat sources in meat and meat-derived products.

Inactivation of Vibrio parahaemolyticus by antimicrobial photodynamic technology using methylene blue.

BACKGROUND: Vibrio parahaemolyticus is the leading causative pathogen of gastroenteritis often related to contaminated seafood. Photodynamic inactivation has been recently proposed as a strategy for killing cells and viruses. The objective of this study was to verify the bactericidal effects caused by photodynamic inactivation using methylene blue (MB) over V. parahaemolyticus via flow cytometry, agarose gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Vibrio parahaemolyticus counts were determined using the most probable number method. A scanning electron microscope and a transmission electron microscope were employed to intuitively analyze internal and external cell structure. RESULTS: Combination of MB and laser treatment significantly inhibited the growth of V. parahaemolyticus. The inactivation rate of V. parahaemolyticus was >99.99% and its counts were reduced by 5 log10 in the presence of 0.05 mg mL(-1) MB when illuminated with visible light (power density 200 mW cm(-2)) for 25 min. All inactivated cells showed morphological changes, leakage of cytoplasm and degradation of protein and DNA. CONCLUSION: Results from this study indicated that photodynamic technology using MB produced significant inactivation of V. parahaemolyticus mainly brought about by the degradation of protein and DNA.

Adverse effects of high glucose levels on somite and limb development in avian embryos.

Gestational diabetes has an adverse impact on fetal musculoskeletal development, but the mechanism involved is still not completely understood. In this study, we investigated the effects of high glucose on the developing somites and their derivate using the chick embryo as a model. We demonstrated that under high glucose, the number of generated somites was reduced and their morphology altered in 2-day old chick embryos. In addition, high glucose repressed the development of the limb buds in 5.5-day old chick embryos. We also demonstrated that high glucose abridged the development of the sclerotome and the cartilage in the developing limb bud. The sonic hedgehog (Shh) gene has been reported to play a crucial role in the development and differentiation of sclerotome. Hence, we examined how Shh expression in the sclerotome was affected under high glucose. We found that high glucose treatment significantly inhibited Shh expression. The high glucose also impaired myotome formation at trunk level - as revealed by immunofluorescent staining with MF20 antibodies. In the neural tube, we established that Wnt3a expression was also significantly repressed. In summary, our study demonstrates that high glucose concentrations impair somite and limb bud development in chick embryos, and suggests that Shh and Wnt genes may play a role in the underlying mechanism.

[Virulence-related genes of Vibrio alginolyticus and its virulence in mice].

OBJECTIVE: We determinated the virulence factors of Vibrio alginolyticus strains isolated from the environment by multiplex PCRs and animal experiments, in order to compare the differences between the highly virulent strain and attenuated virulent strain, and to explore the virulent mechanism of V. alginolyticus in mammals. METHODS: The virulence-related genes of V. alginolyticus were investigated by multiplex PCRs. Hemolysin and pathogenic proteins were detected using Kanagawa phenomenon tests and enzyme activity tests. In vivo pathogenetic tests of V. alginolyticus were done through orogastric and intraperitoneal Kunming mouse. RESULTS: Amylase and lecithinase activities were observed in 100% of the strains, whereas lipase and gelatinase activities were found in only 70% and urease activity was not detected. In Kanagawa phenomenon tests 60% of the strains gave positive results. The related virulence genes such as toxR, Collagenase, tlh, FlaA, ompW, AspA and fur were distributed among 10 strains of V. alginolyticus collected, with the exception of toxS, trh, tdh and UreR. Among those 10 strains, VA009 has shown a strong pathogenesis to the mouse, which caused fluid accumulation and led the mortality rate as high as 80% within 7 days by intraperitoneal infection. CONCLUSION: This study indicates that there is a great difference in pathogenicity among V. alginolyticus strains to mouse. The cell toxicity of V. alginolyticus made more contribution than extracellular secretion, while the extracellular secretion of V. parahaemolyticus played a major role in its toxicity. The virulence gene profiles were consistent between the highly virulent and attenuated virulent strains, indicating that V. alginolyticus might have a different virulence system and different pathogenic mechanism compared with V. parahaemolyticus.

Photodynamic inactivation of methylene blue and tungsten-halogen lamp light against food pathogen Listeria monocytogenes.

The aim of this study was to verify the bactericidal effect and the damage of photodynamic inactivation (PDI) using methylene blue (MB) and tungsten-halogen lamp over Listeria monocytogenes via atomic force microscopy, absorption spectrophotometry, agarose gel electrophoresis, real-time PCR and SDS-PAGE. The obtained data indicated that the viability of L. monocytogenes was ca 7-log reduced by illumination with 10 min tungsten-halogen lamp light under the presence of 0.5 µg mL(-1) MB, and this bactericidal activity against L. monocytogenes of PDI increased proportionally to the concentration of MB and the duration of irradiation. Moreover, after irradiation with MB and visible light, the leakage of intracellular contents was estimated by spectrophotometer at OD(260) and OD(280), which correlated with morphological alterations. Furthermore, genomic DNA cleavage and protein degradation were also detected after PDI treatment. Consequently, breakage of the membrane, damage of the genomic DNA and degradation of bacterial proteins may play an important role in the mechanisms involved in PDI-MB bactericidal activity on L. monocytogenes.

Debittering effect of Actinomucor elegans peptidases on soybean protein hydrolysates.

Effects of the enzymes in Actinomucor elegans extract and the enzyme Alcalase 2.4L on debittering the soybean protein hydrolysates were investigated. When the protein was treated only with the latter, a strong bitterness formed; but it decreased if the protein was treated with both the enzymes. The more the enzymes were used, weaker was the bitterness tasted. SDS-PAGE profile and ESI-MS spectrum of the hydrolysates evidenced that the Alcalase could convert the protein into peptides rapidly, while the enzymes in the A. elegans extract were able to further degrade some peptides which were difficult or unable to be hydrolyzed by the Alcalase. Further systematic analysis of the peptidases showed that the Alcalase exhibited a significant endopeptidase activity towards NBZ-Phe-pNA substrate (p < 0.01), whereas many exopeptidases in the A. elegans extract had the carboxypeptidase activity towards N-CBZ-Ile-Leu (p < 0.01). It is concluded that those exopeptidases presented in the A. elegans extract can benefit by decreasing the bitterness of the soybean protein hydroysate. They are also capable of being used with the Alcalase in a single-step enzymatic reaction to prepare the bitterless protein hydrolysate, which may be an efficient application for food industry.

Simultaneous determination of trilobolide-6-O-isobutyrates A and B in Wedelia trilobata by gas chromatography.

A simple, sensitive, and specific gas chromatographic (GC) method was developed to determine the main bioactive sesquiterpene lactones, trilobolide-6-O-isobutyrates A and B (TBO-A and TBO-B), in Wedelia trilobata, a useful folk herb. A commercially available HP-5 capillary column (30 m x 0.25 mm i.d. x 0.25 microm) was utilized for the direct determination of TBO-A and TBO-B in W. trilobata. Calibration curves were obtained by spiking authentic compounds and the internal standard (ferulic acid) into W. trilobata samples before extraction. Extraction was carried out by refluxing the dried herb (0.5 g) for 1 h in methanol (25 mL). All calibration curves showed good linear regressions (r2 > 0.992) within test ranges. The assay was reproducible and accurate with the overall intraday and interday relative standard deviations and accuracies of less than 10% and more than 90%, respectively. The developed GC method was successfully utilized to analyze the TBO-A and TBO-B in aerial parts and flowers of W. trilobata, indicating that it was suitable for the quality control of this commonly used herb and related traditional Chinese medicines.

Epidemiology of 377 patients with chemical burns in Guangdong province.

A total of 377 patients with chemical burns from all over Guangdong province were admitted to the Guangzhou Red Cross Hospital during the period from January 1987 to December 2001. There were 296 males and 81 females with a male to female ratio of 3.65:1. The mean age of the patients was 26 years. The majority of patients (89.2%) were in the age range of 15-60 years. Professionally, 244 patients (64.7%) were workers, of whom, 232 (95%) of patients were peasant workers. Most of the chemical burns occurred at places away from home (94.4%), especially in the working environment (67.8%). Only 20 patients (5.5%) were injured at home. Chemical burns by accident and by criminal assault were 337 (88.5%) and 40 (10.5%). Strong acids (60.8%), mainly sulfuric acid, nitric acid, hydrofluoric acid, alkali (33.9%), mainly lime and sodium hydroxide were common causative agents. There was a relationship between the incidence of chemical burns and the season, with more patients in July-September and October-December. There were 215 (57.1%) patients who washed the burnsite with water immediately, but the volumes of water and time of washing were not adequate. Patients with total burn surface area (TBSA) of less than 10% comprised the majority of patients (72.7%), with 188 (65.7%) deep partial thickness burns, 116 (40.6%) with full thickness burns, and 60 (21%) with superficial burns. Extremities (lower limb 56.6% and upper limb 51.4%) were the most frequent area of injury. Ocular burns were the most common accompanying injury (14.7%). Operations of autografts and conjunctival flap were carried out on 159 (42.2%) patients. The average period of hospitalization was 22 days. Only 2 (0.7%) deaths occurred in this study. Counter measures to improve this situation must include safety productive education and professional training, use of protective clothing at work, enhancing the concept of legal responsibility, and restricting management and use of corrosive chemicals. Irrigation of the burnsite promptly with substantial volumes of water and an adequately long time will help reduce the morbidity from chemical burns.