Computer-Aided Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase AlyMc.

Alginate lyase degrades alginate by the ß-elimination mechanism to produce unsaturated alginate oligosaccharides (UAOS), which have better bioactivities than saturated AOS. Enhancing the thermal stability of alginate lyases is crucial for their industrial applications. In this study, a feasible and efficient rational design strategy was proposed by combining the computer-aided ΔΔG value calculation with the B-factor analysis. Two thermal stability-enhanced mutants, Q246V and K249V, were obtained by site-directed mutagenesis. Particularly, the t1/2, 50 °C for mutants Q246V and K249V was increased from 2.36 to 3.85 and 3.65 h, respectively. Remarkably, the specific activities of Q246V and K249V were enhanced to 2.41- and 2.96-fold that of alginate lyase AlyMc, respectively. Structural analysis and molecular dynamics simulations suggested that mutations enhanced the hydrogen bond networks and the overall rigidity of the molecular structure. Notably, mutant Q246V exhibited excellent thermal stability among the PL-7 alginate lyase family, especially considering the heightened enzymatic activity. Moreover, the rational design strategy used in this study can effectively improve the thermal stability of enzymes and has important significance in advancing applications of alginate lyase.

Occurrence of N-nitrosodimethylamine in roasted Alaska pollock fillets during processing and storage and preliminary cancer risk assessment.

BACKGROUND: Dried and salt-fermented fish products are important sources of N-nitrosodimethylamine (NDMA) exposure for human. As a potent carcinogen, NDMA was frequently detected in roasted Alaska pollock fillet products (RPFs), which is among the most common fish products in China. Until now, the occurrence and development of NDMA and its precursors (nitrites, nitrates and dimethylamine) in RPFs during processing and storage were not well elucidated, and safety evaluation of this fish product is also urgently needed. RESULTS: The presence of precursors in the raw material was verified and significant increase of nitrates and nitrites during processing was observed. NDMA was found generated during pre-drying (3.7 µg kg-1 dry basis) and roasting (14.6 µg kg-1 dry basis) process. Continuous increase in NDMA content can also be found during storage, especially at higher storage temperature. The 95th percentile of Monte Carlo simulated cancer risk (3.73 × 10-5 ) surpassed the WHO threshold (1.00 × 10-5 ) and sensitivity analysis implies the risk was mainly attributable to NDMA level in RPFs. CONCLUSION: The occurrence of NDMA in RFPs was mainly a result of endogenous factors originating in Alaska pollock during processing and storage rather than exogenous contamination, and temperature played a pivotal role. The preliminary risk assessment results suggest that long-term consumption of RPFs would impose potential health risks for consumers. © 2023 Society of Chemical Industry.

Effects of pegylated recombinant human granulocyte colony-stimulating factor on lymphocytes and white blood cells of patients with malignant tumor.

We investigated the effect of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) on lymphocytes and white blood cells of patients with malignant tumors. After PEG-rhG-CSF treatment, the count of lymphocytes increased in 66 cases, remained unchanged in 2 cases, and decreased in 20 cases. The difference in lymphocyte count before and after treatment was statistically significant (P < 0.001). White blood cell changes were positively correlated with lymphocyte changes (r = 0.36, P = 0.001). In the subgroup with increased white blood cells (n = 80), there were 62 cases with increased lymphocytes, 1 case with unchanged lymphocytes, and 17 cases with decreased lymphocytes after PEG-rhG-CSF treatment. There was significant difference in the count of lymphocytes and white blood cells (P < 0.001). In the subgroup with 6 mg of PEG-rhG-CSF (n = 66) and the subgroup with 3 mg of PEG-rhG-CSF (n = 22), the changes of white blood cell and lymphocyte counts before and after treatment were statistically significant (P < 0.001). The two were positively correlated in the 6 mg PEG-rhG-CSF subgroup, with correlation coefficient r = 0.34 (P = 0.002). PEG-rhG-CSF can increase the count of lymphocytes and white blood cells in patients with malignant tumors, and the increase of lymphocytes is positively correlated with the increase of white blood cells.

Effect of drying processes on the occurrence of lipid oxidation-derived 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal in Spanish mackerel (Scomberomorus niphonius).

In this study, dry-cured Spanish mackerel (Scomberomorus niphonius, DCSM) was prepared via three different methods (hot-air drying, cold-air drying, and sun drying). The content of 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) derived from lipid oxidation in whole processes was investigated by HPLC-MS/MS. The changes in fatty acid composition were detected by GC-MS, and the degree of lipid oxidation was evaluated by the levels of acid values (AV), peroxide values (POV), and thiobarbituric acid-reactive substances (TBARS). The results showed that the drying process significantly accelerated lipid oxidation in DCSM. The contents of HHE and HNE were significantly increased after processing. The content of HHE was higher by 18.44-, 13.45-, and 16.32-folds compared with that of HNE after three different processes, respectively. The HHE and HNE contents fluctuated upward during the hot-air and cold-air drying process. However, the contents of HHE and HNE increased time-dependent during the sun drying process, with the highest values of 86.33 ± 10.54 and 5.29 ± 0.54 mg/kg fish among the three different processes. Besides, there was a significant positive correlation between HHE contents and n-3 fatty acids content in hot-air drying and sun drying processes (Pearson's r = .991/.996), and HNE occurrence was closely related to n-6 fatty acid content in sun drying process (Pearson's r = .989). Regression analysis indicated that the content of HHE and TOTOXTBA values in DCSM showed good linear relationships (R 2 value = .907), which suggested that the content of HHE could be used to estimate the oxidative deterioration of dry-cured fish products.

Gut Microbiota and Transcriptomics Reveal the Effect of Human Norovirus Bioaccumulation on Oysters (Crassostrea gigas).

Human norovirus (HuNoV) is a major foodborne pathogen that causes acute viral gastroenteritis, and oysters are one of the main carriers of HuNoV transmission. While progress has been made toward understanding the pattern of oyster-bioaccumulated HuNoV, the response of oysters to HuNoV bioaccumulation, including changes in gene expression and gut microbiota, is unclear. In this study, histo-blood group antigen (HBGA)-like molecule expression and gene regulation features and the HuNoV-microbiome interactions of oysters during HuNoV bioaccumulation were characterized. With the prolongation of bioaccumulation time, the HuNoV content and expression of type A HBGA-like molecules in oysters increased and stabilized. HuNoV also altered the expression of immunity- and glycosphingolipid biosynthesis-related genes. Prolonged bioaccumulation of HuNoV can reduce the abundance and change the composition of the oyster gut microbiota. In particular, with the extension of bioaccumulation time, the abundance of Blautia, Agathobacter, Faecalibacterium, Terrisporobacter, Bifidobacterium, Lactobacillus, and Ruminococcus decreased, while the abundance of Vibrio and Alphaproteobacteria increased. This study provides potential candidates for identifying functional genes involved in the bioaccumulation of HuNoV in oysters. More importantly, it provides the first description of the changes in gut microbiota during HuNoV bioaccumulation in oysters. IMPORTANCE The role of the oyster gut microbiota in HuNoV bioaccumulation is poorly understood. This study revealed, for the first time, the changes in gut microbiota and gene expression of oysters with HuNoV bioaccumulation. This study enriches the understanding of the impact of HuNoV bioaccumulation on oysters and provides a new direction for the study of the molecular mechanism of HuNoV bioaccumulation in oysters.

Contamination, bioaccumulation mechanism, detection, and control of human norovirus in bivalve shellfish: A review.

Human norovirus (HuNoV) is a major foodborne pathogen that causes acute viral gastroenteritis, and bivalve shellfish are one of the main carriers of HuNoV transmission. A comprehensive understanding of bivalve shellfish-related HuNoV outbreaks focusing on contamination factors, bioaccumulation mechanisms, and pre- and post-harvest interventions is essential for the development of effective strategies to prevent contamination of shellfish. This review comprehensively surveys the current knowledge on global contamination and non-thermal treatment of HuNoV in bivalve shellfish. HuNoV contamination in bivalve shellfish is significantly related to the season and water. While evaluating the water quality of shellfish-inhabited waters is a key intervention, the development of non-heat treatment technology to effectively inactivate the HuNoV in bivalve shellfish while maintaining the flavor and nutrition of the shellfish is also an important direction for further research. Additionally, this review explores the bioaccumulation mechanisms of HuNoV in bivalve shellfish, especially the mechanism underlying the binding of histo-blood group antigen-like molecules and HuNoV. The detection methods for infectious HuNoV are also discussed. The establishment of effective methods to rapidly detect infectious HuNoV and development of biological components to inactivate or prevent HuNoV contamination in shellfish also need to be studied further.

Physicochemical and Fibril Formation Properties of Pufferfish (Takifugu obscurus) Skin Collagen from Solvent Extraction in Different Conditions.

Acid-solubilized (ASC) and pepsin-solubilized collagen (PSC) extracted at 4 °C (ASC-4 and PSC-4), 12 °C (ASC-12 and PSC-12), and 20 °C (ASC-20 and PSC-20) from the skin of farmed pufferfish (Takifugu obscurus) was characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier-transform infrared spectroscopy (FTIR), and fibril-forming tests. The results indicate that extraction at 12 °C can effectively improve the extraction efficiency of natural collagen compared with extraction at 4 °C. However, extraction at 20 °C results in a decrease in molecular integrity, thus, inducing the resultant collagen to degrade or even lose fibril-forming ability. Transmission electron microscope (TEM) images revealed that ASC-4, PSC-4, ASC-12, and PSC-12 can assemble into fibrils with D-periodicities, and ASC-20 associated into molecular aggregates alongside partial D-banded fibrils, while no well-defined fibrils were observed in PSC-20. Scanning electron microscope (SEM) analysis confirmed the well-defined fibril morphologies of ASC-4, PSC-4, ASC-12, and PSC-12 with imino acid contents between 190.0 and 197.8 residues/1000 residues. The denaturation temperature of ASC-4, PSC-4, ASC-12 and PSC-12 was 30.0, 27.6, 25.9 and 22.7 °C, respectively. This study indicates that ASC and PSC extracted at 4 °C and 12 °C could be alternatives to terrestrial collagens for industrial applications.

Preliminary Quantitative Risk Assessment of Norovirus in Shellfish in the Yellow Sea and Bohai Sea of China.

Norovirus (NoV) is a main foodborne pathogen of acute gastroenteritis in the world. A preliminary quantitative risk assessment (QRA) was conducted to evaluate the health risk caused by this virus in shellfish in the Yellow Sea and Bohai Sea of China. The QRA framework was established from the process of shellfish at retail through cooking at home to consumer consumption. The prevalence and quantity of NoVs in shellfish, cooking methods, internal temperature and time of shellfish in different cooking conditions, shellfish consumption frequency, and consumption amount were analyzed in the exposure assessment. The results of exposure assessment were introduced into the beta-Poisson dose-response model, and Monte Carlo analysis was used to calculate the risk of gastroenteritis caused by shellfish consumption in the cities around the Yellow Sea and Bohai Sea of China. The results showed that the probability of illness caused by NoVs due to shellfish consumption per year (Pill,yr) was 1.86 × 10-5. It was estimated that the annual number of patients with gastroenteritis per 1,000,000 general population (Nexp,mil) was 0.10, 1.23, 16.90, and 0.38 for population aged 0-4, 5-18, 19-64, and >65 years, respectively. This assessment provides valuable information such as the probability of illness associated with the consumption of shellfish and it also provides a reference for further in-depth QRA of NoVs in shellfish or other foods.

Research progress on applications of calcium derived from marine organisms.

Calcium is an important mineral that plays an integral role in human health, especially bone health. Marine biological calcium is an abundant resource that is generally accepted and has a complex active structure. This review evaluates research progress on marine biological calcium with regards to its sources, use of calcium supplements, calcium bioavailability, and novel applications of marine calcium. The potential for future development and the use of products incorporating marine biological calcium in biomedical research and the pharmaceutical, health care, and food industries are also reviewed. The goal of this review is to provide a comprehensive documentation on resource utilization and product development from marine organisms.

Comparison of Physicochemical Characteristics and Fibril Formation Ability of Collagens Extracted from the Skin of Farmed River Puffer (Takifugu obscurus) and Tiger Puffer (Takifugu rubripes).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of river puffer (ASC-RP and PSC-RP) and tiger puffer (ASC-TP and PSC-TP) were extracted and physicochemically examined. Denaturation temperature (Td) for all the collagens was found to be 25.5-29.5 °C, which was lower than that of calf skin collagen (35.9 °C). Electrophoretic patterns indicated all four samples were type I collagen with molecular form of (α1)2α2. FTIR spectra confirmed the extracted collagens had a triple-helical structure, and that the degree of hydrogen bonding in ASC was higher than PSC. All the extracted collagens could aggregate into fibrils with D-periodicity. The fibril formation rate of ASC-RP and PSC-RP was slightly higher than ASC-TP and PSC-TP. Turbidity analysis revealed an increase in fibril formation rate when adding a low concentration of NaCl (less than 300 mM). The fibril formation ability was suppressed with further increasing of NaCl concentration, as illustrated by a reduction in the turbidity and formation degree. SEM analysis confirmed the well-formed interwoven structure of collagen fibrils after 24 h of incubation. Summarizing the experimental results suggested that the extracted collagens from the skin of river puffer and tiger puffer could be considered a viable substitute to mammalian-derived collagens for further use in biomaterial applications.

Presence and Distribution of Histo-Blood Group Antigens in Pacific Oysters and the Effects of Exposure to Noroviruses GI.3 and GII.4 on Their Expression.

Noroviruses (NoVs) are one of the most important foodborne viral pathogens worldwide. Oysters are common carriers of NoVs and are responsible for their transmission. NoVs recognize human histo-blood group antigens (HBGAs) as receptors. Recent studies indicate that HBGA-like molecules also exist in oyster tissues and that they may play a key role in the binding of NoVs. However, the mechanism by which different genotypes of NoV accumulate in different oyster tissues is unknown. In this study, the presence and distribution of different types of HBGA-like molecules were evaluated in 240 oysters collected from the Shandong Peninsula of People's Republic of China for 1 year. The HBGA-like molecules were detected at various rates and expressed at different levels in different tissues. Immunohistochemistry confirmed the diversity of HBGA-like molecules in four oyster tissues. Eight types of HBGA-like molecules (types A, B, H1, Lewis x, Lewis y, Lewis a, Lewis b, and precursor) were assessed in different tissues. Of these, the type A HBGA-like molecule was consistently expressed in the gills, digestive tissue, and mantle, while types H1 and Lewis b HBGA-like molecules were expressed in the digestive tissues. The expression of HBGA-like molecules in response to the NoV challenge was investigated. The levels of types A, H1, and Lewis x increased significantly in specific oyster tissues after exposure to genogroup II, genotype 4 (GII.4) or genogroup I, genotype 3 (GI.3) NoV. The real-time reverse transcription PCR assays indicated that GI.3 NoV mainly accumulated in the digestive tissues of oysters, whereas GII.4 NoV accumulated in the gills, mantle, and digestive tissues. These results provide new insights into the mechanism of NoV bioaccumulation in oysters and suggest that NoV accumulation in oysters may be related to the expression of HBGA-like molecules.

iTRAQ-based quantitative proteomics reveals the biochemical mechanism of cold stress adaption of razor clam during controlled freezing-point storage.

Razor clam is a major cultivated shellfish of great economic importance and high nutritional value. Due to high corruptible potential, razor clam is generally preserved by controlled freezing-point storage (CFPS). Here, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling to investigate the biochemical mechanism of cold stress adaption in razor clam during CFPS. In total, 369 proteins were quantified, and 27 of them were identified as differentially expressed proteins during CFPS, mostly involved in energy metabolism process, DNA duplication and protein synthesis, and stress response, specifically, MAPK is the predominant pathway. Further qPCR results revealed H2A and S6K 2 alpha to be the critical post-transcriptionally regulated genes. Our results provided proteomics information with respect to the biochemical mechanism of cold stress adaption in razor clam, shed light on the further elongation of razor clams storage period, and help clarify the novel mechanisms of cold tolerance.

Norovirus contamination and the glycosphingolipid biosynthesis pathway in Pacific oyster: A transcriptomics study.

Noroviruses are the primary pathogens associated with shellfish-borne gastroenteritis outbreaks. These viruses remain stable in oysters, suggesting an active mechanism for virus concentration. In this study, a deep RNA sequencing technique was used to analyze the transcriptome profiles of Pacific oysters at different time points after inoculation with norovirus (GII.4). We obtained a maximum of 65, 294, 698 clean sample reads. When aligned to the reference genome, the average mapping ratio of clean data was approximately 65%. In the samples harvested at 12, 24, and 48 h after contamination, 2,223, 2,990, and 2020 genes, respectively, were differentially expressed in contaminated and non-contaminated oyster digestive tissues, including 500, 1748, and 1039 up-regulated and 1723, 1242, and 981 down-regulated genes, respectively. In particular, FUT2 and B3GNT4, genes encoding the signaling components of glycosphingolipid biosynthesis, were significantly up-regulated in contaminated samples. In addition, we found up-regulation of some immune- and disease-related genes in the MHC I pathway (PA28, HSP 70, HSP90, CANX, BRp57, and CALR) and MHC II pathway (GILT, CTSBLS, RFX, and NFY), although NoVs did not cause diseases in the oysters. We detected two types of HBGA-like molecules with positive-to-negative ratios similar to type A and H1 HBGA-like molecules in digestive tissues that were significantly higher in norovirus-contaminated than in non-contaminated oysters. Thus, our transcriptome data analysis indicated that a human pathogen (GII.4 Norovirus) was likely concentrated in the digestive tissues of oysters via HBGA-like molecules that were synthesized by the glycosphingolipid biosynthesis pathway. The identified differentially expressed genes also provide potential candidates for functional analysis to identify genes involved in the accumulation of noroviruses in oysters.

[Gene deletion and functional analysis of the heptyl glycosyltransferase (waaF) gene in Vibrio parahemolyticus O-antigen cluster].

OBJECTIVE: To construct heptyl glycosyltransferase gene II (waaF) gene deletion mutant of Vibrio parahaemolyticus, and explore the function of the waaF gene in Vibrio parahaemolyticus. METHODS: The waaF gene deletion mutant was constructed by chitin-based transformation technology using clinical isolates, and then the growth rate, morphology and serotypes were identified. The different sources (O3, O5 and O10) waaF gene complementations were constructed through E. coli S17λpir strains conjugative transferring with Vibrio parahaemolyticus, and the function of the waaF gene was further verified by serotypes. RESULTS: The waaF gene deletion mutant strain was successfully constructed and it grew normally. The growth rate and morphology of mutant were similar with the wild type strains (WT), but the mutant could not occurred agglutination reaction with O antisera. The O3 and O5 sources waaF gene complementations occurred agglutination reaction with O antisera, but the O10 sources waaF gene complementations was not. CONCLUSION: The waaF gene was related with O-antigen synthesis and it was the key gene of O-antigen synthesis pathway in Vibrio parahaemolyticus. The function of different sources waaF gene were not the same.

Characterization and toxicology evaluation of chitosan nanoparticles on the embryonic development of zebrafish, Danio rerio.

In the present study, chitosan nanoparticles were prepared, characterized and used to evaluate the embryonic toxicology on zebrafish (Danio rerio). The average particle size of chitosan nanoparticles was 84.86nm. The increased mortality and decreased hatching rate was found in the zebrafish embryo exposure to normal chitosan particles and chitosan nanoparticles with the increased addition concentration. At 120h post-fertilization (hpf), the rate of mortality were 25.0 and 44.4% in the groups treated with chitosan nanoparticles and normal chitosan particles at 250mg/L, respectively. At 72hpf, the hatching rate in the groups treated with normal chitosan particles were lower (P<0.01) at 300 and 400mg/L than those of the corresponding control groups, respectively. However, there were no significant differences between the groups treated with chitosan nanoparticles and the control groups across all the addition concentrations. More abundant typical malformation of embryos was observed in the groups treated with normal chitosan particles compared with those treated with chitosan nanoparticles. The LC50 (medium lethal concentration) of chitosan nanoparticles was 280mg/L at 96hpf and 270mg/L at 120hpf. As for normal chitosan particles, the LC50 was 257mg/L at both 96hpf and 120hpf. The TC50 (medium teratogenic concentration) of the zebrafish treated with chitosan nanoparticles and normal chitosan particles were 257mg/L and 137mg/L, respectively. It indicated that the chitosan nanoparticles were relatively more secure compared with normal chitosan particles.

[Research Progress in Norovirus Bioaccumulation in Shellfish].

Noroviruses (NoVs) are one of the most important foodborne viral pathogens worldwide. Shellfish are the most common carriers of NoVs as they can concentrate and accumulate large amounts of the virus through filter feeding from seawater. Shellfish may selectively accumulate NoVs with different genotypes, and this bioaccumulation may depend on the season and location. Our previous studies found various histo-blood group antigens (HBGAs) in shellfish tissues. While HBGAs might be the main reason that NoVs are accumulated in shellfish, the detailed mechanism behind NoV concentration and bioaccumulation in shellfish is not clear. Here we review current research into NoV bioaccumulation, tissue distribution, seasonal variation, and binding mechanism in shellfish. This paper may provide insight into controlling NoV transmission and decreasing the risks associated with shellfish consumption.

Distribution of Marine Lipophilic Toxins in Shellfish Products Collected from the Chinese Market.

To investigate the prevalence of lipophilic marine biotoxins in shellfish from the Chinese market, we used hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure levels of okadaic acid (OA), azaspiracid (AZA1), pectenotoxin (PTX2), gymnodimine (GYM), and spirolide (SPX1). We collected and analyzed 291 shellfish samples from main production sites along a wide latitudinal transect along the Chinese coastline from December 2008 to December 2009. Results revealed a patchy distribution of the five toxins and highlighted the specific geographical distribution and seasonal and species variation of the putative toxigenic organisms. All five lipophilic marine biotoxins were found in shellfish samples. The highest concentrations of OA, AZA1, PTX2, GYM, and SPX1 were 37.3, 5.90, 16.4, 14.4, and 8.97 µg/kg, respectively. These values were much lower than the legislation limits for lipophilic shellfish toxins. However, the value might be significantly underestimated for the limited detection toxins. Also, these toxins were found in most coastal areas of China and were present in almost all seasons of the year. Thus, these five toxins represent a potential threat to human health. Consequently, studies should be conducted and measures should be taken to ensure the safety of the harvested product.

Effect of Ursolic Acid on Breast Cancer Resistance Protein-mediated Transport of Rosuvastatin In Vivo and Vitro.

OBJECTIVE: To evaluate whether ursolic acid can inhibit breast cancer resistance protein (BCRP)-mediated transport of rosuvastatin in vivo and in vitro. METHODS: Firstly, we explored the pharmacokinetics of 5-fluorouracil (5-FU, a substrate of BCRP) in rats in the presence or absence of ursolic acid. Secondly, we studied the pharmacokinetics of rosuvastatin in rats in the presence or absence of ursolic acid or Ko143 (inhibitor of BCRP). Finially, the concentration-dependent transport of rosuvastatin and the inhibitory effects of ursolic acid and Ko143 were examined in Madin-Darby Canine Kidney (MDCK) 2-BCRP421CC (wild type) cells and MDCK2-BCRP421AA (mutant type) cells. RESULTS: As a result, significant changes in pharmacokinetics parameters of 5-FU were observed in rats following pretreatment with ursolic acid. Both ursolic acid and Ko143 could significantly affect the pharmacokinetics of rosuvastatin. The rosuvastatin transport in the BCRP overexpressing system was increased in a concentration-dependent manner. However, there was no statistical difference in BCRP-mediated transport of rosuvastatin betweent the wild type cells and mutant cells. The same as Ko143, ursolic acid inhibited BCRP-mediated transport of rosuvastatin in vitro. CONCLUSION: Ursolic acid appears to be a potent modulator of BCRP that affects the pharmacokinetic of rosuvastatin in vivo and inhibits the transport of rosuvastatin in vitro.

The presence of genogroup II norovirus in retail shellfish from seven coastal cities in China.

Noroviruses (NoVs) are commonly occurring pathogens that cause gastroenteritis. Outbreaks of viral diseases have often been ascribed to the consumption of contaminated shellfish. Our objective was to evaluate the presence and contamination levels of NoV in shellfish sold at seafood markets in China. We tested 840 shellfish samples (Crassostrea gigas, Mytilus edulis, Azumapecten farreri, SinoNoVacula constricta, Scapharca subcrenata, Ruditapes philippinarum) that were collected from seven cities around the Yellow and Bohai Seas in China between December 2009 and November 2011. We used real-time RT-PCR to detect NoV in purified concentrates from the stomach and digestive diverticula of these shellfish. NoV was detected in 19.35 % (N = 155), 16.67 % (N = 114), 5.70 % (N = 158), 8.82 % (N = 136), 13.74 % (N = 131), and 16.44 % (N = 146) of oyster, mussel, scallop, razor clam, ark shell, and clam samples, respectively. The average detection rate was 13.33 % (112/840). Nucleotide sequencing of the NoV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.12, except two that belonged to GI.3. More than 10² copies of the NoV genome were detected in 69 of 112 positive shellfish samples. Our results suggest that ~13 % of shellfish harbor NoV, and GII.12 NoV is the primary strain in shellfish purchased at markets in seven coastal cities in China.

[Determination of azaspiracid-1 in shellfishes by liquid chromatography with tandem mass spectrometry].

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of azaspiracid-1 (AZA1) in shellfishes was described. After being extracted using methanol and water (80:20, v/v), the extract was cleaned-up by solid phase extraction (SPE) of MAX column, then determined by using a reversed-phase high performance liquid chromatography (HPLC) isocratic program coupled with tandem mass spectrometry in selected reaction monitoring mode (SRM). And the extract was eluted with acetonitrile-water (80:20, v/v) on an Atlantis dC18 column (150 mm x 4.6 mm, 5.0 microm) with mobile phase containing 50 mmol/L formic acid and 2 mmol/L ammonium formate. The detection limit was 11.00 pg/g. The calibration curve was linear (R2 = 0.998 1) in the range of 48.85-2 442 ng/L. The average recoveries of the shellfish tissue extract at three spiked levels (36.64, 73.27, 146.54 pg/g) were from 75.8% to 82.5% (n = 6). The relative standard derivations (RSDs) were less than 10%. The 112 shellfish samples from the local markets of Dalian, Qingdao, Guangzhou were detected by the method, and AZA1 was detected in some samples from Dalian and Guangzhou. The results showed that the method is simple, rapid, sensitive and suitable for the detection of AZA1 in shellfishes.