Detection of GI and GII noroviruses in drinking water and vegetables using filtration and real-time RT-PCR

The purpose of the study was to provide a rapid and sensitive method for detecting NoV GI and NoV GII in drinking water and vegetables. The method is based on viral concentration by microporous membrane adsorption method before RNA extraction and real-time RT-PCR amplification. Then water and vegetable samples which artificially contaminated with NoV GI and GII stool samples were used to determine the mean virus recoveries and the method sensitivity. The method showed the detection limit of NoV GI was 4.13 × 10² copies/500 mL for drinking water and 4.13 × 10³ copies/15 g for lettuce and coriander. The detection limit of NoV GII was 2.94 × 10¹ copies/500 mL for distilled water, 2.94 × 10² copies/500 mL for Mountain spring water and mineral water, and 2.94 × 10³ copies/15 g for lettuce and coriander. The method described provides a valuable tool for monitoring the potential public health risks associated with noroviruses contamination in drinking water and vegetables.     

Multicenter Collaborative Trial Evaluation of a Method for Detection of Human Adenoviruses in Berry Fruit

The qualitative performance characteristics of a qPCR-based method to detect human adenoviruses in raspberries were determined through a collaborative trial involving 11 European laboratories. The method incorporated a sample process control (murine norovirus) and an internal amplification control. Trial sensitivity or correct identification of 25-g raspberry samples artificially contaminated with between 5?×?10² and 5?×?10⁴?PFU was 98.5%; the accordance and concordance were 97.0%. The positive predictive value was 94.2%. The trial specificity or percentage correct identification of non-artificially contaminated samples was 69.7%; the accordance was 80.0% and the concordance was 61.7%. The negative predictive value was 100%. Application of a method for the detection of human adenoviruses in food samples could be useful for routine monitoring for food safety management. It would help to determine if a route of contamination exists from human source to food supply chain which pathogenic viruses such as norovirus and hepatitis A virus could follow.     

Simultaneous recovery of bacteria and viruses from contaminated water and spinach by a filtration method

Water and leafy vegetables eaten fresh are increasingly reported as being involved in food-borne illness cases. The pathogenic agents responsible for these infections are mainly bacteria and viruses and are present in very small quantities on the contaminated food matrices. Laboratory techniques used to isolate or detect the contaminating agent differ enormously according to the type of microorganisms, generating time and economical losses. The purpose of this study was to optimize a single method which allows at the same time the recovery and concentration of these two main types of pathogenic organisms. Water and spinach samples were artificially contaminated with the feline calicivirus (FCV), rotavirus, hepatitis A virus (HAV), Escherichia coli, Listeria monocytogenes, Campylobacter jejuni and Salmonella Typhimurium. The principle behind the recovery technique is based on the use of a positively charged membrane which adsorbs both viruses and bacteria present in the water or in the rinse from the vegetables. Using conventional microbiology, PCR and RT-PCR, this filtration technique allowed a detection level superior to 10² CFU/g for S. Typhimurium, E. coli, L. monocytogenes and C. jejuni and to 10¹ PFU/g for FCV, HAV and rotavirus. This combined method can also be applied to other bacterial and viral species for the identification of the responsible agent for food-borne illnesses.     

Aqueous matrix compositions and pH influence feline calicivirus inactivation by high pressure processing

The individual effects of pH (pH 3 to 8), NaCl (0 to 21%), sucrose (0 to 70%), and whey protein (0 to 2%) on pressure resistance of feline calicivirus (FCV) in Dulbecco's modified Eagle medium with 10% fetal bovine serum were determined. At pH 3 through 8, the virus was more resistant to pressure at a pH of < or = 5.2. For FCV samples with sucrose (up to 40%) or NaCl (up to 12%), the amount of FCV inactivated by pressure was inversely proportional to the sucrose or NaCl concentration. For example, a treatment of 250 MPa at 20 degrees C for 5 min reduced the FCV titer by 5.1 log PFU/ml without added sucrose and by 0.9 log PFU/ml with 40% sucrose. Reduced pressure sensitivity with increasing NaCl and sucrose concentrations was not a simple function of water activity. Different PFU reductions were observed for NaCl and sucrose samples with equivalent water activity. When protein at concentrations up to 2% did not provide a protective effect. The combined effect of NaCl and sucrose at 4 and 20 degrees C on pressure resistance of FCV also was examined. When both NaCl and sucrose were added to the FCV stock, they had an additive effect on increasing the pressure resistance of FCV. The individual (6% NaCl or 20% sucrose) and combined (6% NaCl plus 20% sucrose) resistance effects did not abrogate enhanced inactivation for pressure treatments at 4 degrees C compared with those at 20 degrees C. Aqueous matrix compositions, in particular different concentrations of NaCl and sucrose or different pH values, can substantially alter the efficiency of virus inactivation by high pressure processing.     

Persistence of caliciviruses on environmental surfaces and their transfer to food

The noroviruses (NoV) are a common cause of human gastroenteritis whose transmission by foodborne routes is well documented. Fecally contaminated surfaces are likely to contribute to this foodborne transmission and to the propagation of viral disease outbreaks. The purpose of this study was to (i) investigate the stability of NoV on various food preparation surfaces; and (ii) evaluate the degree of virus transfer from these surfaces to a model-ready-to-eat (RTE) food. For the virus persistence experiments, stainless steel, formica and ceramic coupons were artificially contaminated with Norwalk virus (NV), the prototype genogroup I NoV; NV RNA; or feline calicivirus (FCV) F9 (a NoV surrogate), stored at ambient temperature for up to 7 d, and periodically assayed for detection. In the transfer experiments, stainless steel coupons were inoculated with NV or FCV F9 and allowed to dry for 10, 30 and 60 min, after which lettuce leaves were exposed to the surface of the coupons at various contact pressures (10, 100, and 1000 g/9 cm2). Virus recovery was evaluated by RT-PCR (for NV and NV RNA) or by plaque assay (for FCV F9) using Crandell Reese Feline Kidney (CRFK) cells. NV and FCV were detected on all three surfaces for up to 7 d post-inoculation; for FCV, there was an approximate 6 to 7-log10 drop in virus titer over the 7 d evaluation period. By contrast, when stainless steel was inoculated with purified NV RNA, RT-PCR detection was not possible beyond 24 h. Transfer of both NV and FCV from stainless steel surfaces to lettuce occurred with relative ease. This study confirms lengthy NoV persistence on common food preparation surfaces and their ease of transfer, confirming a potential role for environmental contamination in the propagation of viral gastroenteritis.     

Plasmonic ELISA based on DNA-directed gold nanoparticle growth for Cronobacter detection in powdered infant formula samples

The traditional gold nanoparticle (AuNP) growth-based plasmonic ELISA (pELISA) strictly and directly controlled by reducing reagents can achieve high sensitivity, but it remains fragile toward the surrounding environment. This work developed a sandwich pELISA for Cronobacter detection in powdered infant formula samples by mediating AuNP growth through DNA. In this assay, DNA adsorbed on the surface of gold nanoseeds guided the anisotropic crystal growth with hydroxylamine as a reducing reagent, and the catalase–hydrogen peroxide (Cat–H₂O₂) system was introduced to bridge the DNA-directed AuNP growth and pELISA, as such DNA can be cleaved into fragments by the hydroxyl radical generated from oxidation of H₂O₂ through Fenton reagents. Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (Cronobacter muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 10⁵ cfu/mL. This method also revealed a good linear range (3 × 10² to 3 × 10⁷ cfu/mL) for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection of 1.6 × 10² cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase-based conventional ELISA (2.6 × 10⁴ cfu/mL). By taking advantage of highly stable DNA-directed AuNP growth, the proposed method shows a good performance in powdered infant formula samples spiked with different concentrations of C. muytjensii ATCC 51329 with average recoveries ranging from 90.79 to 119.09% and coefficient of variation ranging from 4.24 to 9.55%. These values corresponded to an acceptable accuracy and precision for the proposed method. In brief, this work shows potential for screening other analytes in food safety, clinical diagnostics, and environmental monitoring.     

Fate of Listeria on various food contact and noncontact surfaces when treated with bacteriophage

Study objective was to determine efficacy of a bacteriophage suspension against Listeria spp. when applied to three common types of materials used in food manufacturing facilities. Materials included two food contact materials (stainless steel and polyurethane thermoplastic belting) and one noncontact material (epoxy flooring). Coupons of each material were inoculated with a cocktail containing L. monocytogenes and L. innocua (4 to 5-log₁₀ CFU/cm²). Two phage concentrations and a control, 0, 2 × 10⁷ and 1 × 10⁸ PFU/cm² were evaluated. Treated samples were held at 4 or 20°C for 1 and 3 hr to determine the effect of temperature and treatment time. Reductions in Listeria populations ranged from 1.27 to 3.33 log₁₀ CFU/cm² on stainless steel, from 1.17 to 2.76 log₁₀ CFU/cm² on polyurethane thermoplastic belting, and from 1.19 to 1.76 log₁₀ CFU/cm² on epoxy resin flooring. Higher phage concentration (1 × 10⁸ PFU/cm²), longer treatment time (3 hr), and processing area temperature of 20°C showed a greater (p ≤ .05) reduction of Listeria on the stainless-steel and polyurethane thermoplastic belting coupons. Overall, Listeria reduction by phage treatment occurred on all three materials tested, under all conditions.     

A simple method to recover Norovirus from fresh produce with large sample size by using histo-blood group antigen-conjugated to magnetic beads in a recirculating affinity magnetic separation system (RCAMS)

Human norovirus (NoV) outbreaks are major food safety concerns. The virus has to be concentrated from food samples in order to be detected. PEG precipitation is the most common method to recover the virus. Recently, histo-blood group antigens (HBGA) have been recognized as receptors for human NoV, and have been utilized as an alternative method to concentrate human NoV for samples up to 40 mL in volume. However, to wash off the virus from contaminated fresh food samples, at least 250 mL of wash volume is required. Recirculating affinity magnetic separation system (RCAMS) has been tried by others to concentrate human NoV from large-volume samples and failed to yield consistent results with the standard procedure of 30 min of recirculation at the default flow rate. Our work here demonstrates that proper recirculation time and flow rate are key factors for success in using the RCAMS. The bead recovery rate was increased from 28% to 47%, 67% and 90% when recirculation times were extended from 30 min to 60 min, 120 min and 180 min, respectively. The kinetics study suggests that at least 120 min recirculation is required to obtain a good recovery of NoV. In addition, different binding and elution conditions were compared for releasing NoV from inoculated lettuce. Phosphate-buffered saline (PBS) and water results in similar efficacy for virus release, but the released virus does not bind to RCAMS effectively unless pH was adjusted to acidic. Either citrate-buffered saline (CBS) wash, or water wash followed by CBS adjustment, resulted in an enhanced recovery of virus. We also demonstrated that the standard curve generated from viral RNA extracted from serially-diluted virus samples is more accurate for quantitative analysis than standard curves generated from serially-diluted plasmid DNA or transcribed-RNA templates, both of which tend to overestimate the concentration power. The efficacy of recovery of NoV from produce using RCAMS was directly compared with that of the PEG method in NoV inoculated lettuce. 40, 4, 0.4, and 0.04 RTU can be detected by both methods. At 0.004 RTU, NoV was detectable in all three samples concentrated by the RCAMS method, while none could be detected by the PEG precipitation method. RCAMS is a simple and rapid method that is more sensitive than conventional methods for recovery of NoV from food samples with a large sample size. In addition, the RTU value detected through RCAMS-processed samples is more biologically relevant.     

A Rapid and Visual Single Primer Isothermal Amplification-Based Method for the Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Raw Pork Products

Staphylococcus aureus (S. aureus) is an important food-borne pathogen which poses a severe threat to public health worldwide. Rapid detection of S. aureus with high sensitivity is of particular importance for food safety. In this study, a novel single primer isothermal amplification (SPIA) method was established to detect S. aureus in food, targeting the accessory gene regulator (agr) gene with a DNA/RNA primer. The developed SPIA method has the advantages of visualization and avoiding tedious electrophoresis. In order to confirm the specificity of this method, 7 S. aureus strains and 26 non-S. aureus strains were detected with their pure cultures. The sensitivity and detection limit of S. aureus with artificially inoculated raw pork products by SPIA were evaluated through fluorescence and turbidity by naked eye and the amplification curve, which were 4.3?×?10⁰ CFU/mL and 5.6?×?10⁰ CFU/g, respectively. Compared with the conventional PCR method, the SPIA has 100-fold higher sensitivity and 100-fold lower detection limit. Therefore, the developed SPIA method is a potentially reliable tool for rapid and visual detection of S. aureus in food.     

Practical application of bacteriophage in food manufacturing facilities for the control of Listeria sp.

Listeria monocytogenes is a foodborne pathogen with the ability to persist and form biofilm matrices in processing environments of food manufacturing facilities. Bacteriophages are bacterial viruses with host specific lethality. Published research on the application of phage to control Listeria sp. in manufacturing environments is limited. In this study, we have assessed the capacity of bacteriophage P100 (Listex™) to reduce incidence of Listeria sp. in the ready-to-eat (RTE) environment of refrigerated (4°C) and ambient (20°C) temperature facilities using two different application strategies. A moderate application applied as a single treatment every 24 hr over three days (2 × 10⁷ PFU/ml) and an intensified application applied once every 6 hr over a 24 hr period (1 × 10⁸ PFU/ml). Environmental nonfood contact surface (NFCS) samples were collected and analyzed for the presence of Listeria sp. before and after treatment. When the moderate treatment protocol was applied the incidence of positives decreased from 51.3 to 17.5% in the 4°C environment and from 67.5 to 23.1% in the 20°C production area. For the intensified phage treatment method, the initial positive rate in the 4°C environment ranged from 5 to 47.5%, with an overall 43% reduction in Listeria sp. In the 20°C facility, initial environmental Listeria sp. ranged from 15 to 50%, with an overall reduction of 32% after treatment with phage P100. Data indicate the application of Listeria specific phage P100 in RTE food production environments by either the moderate or intensified application method can reduce incidence and be considered an additional intervention strategy for controlling this pathogen on NFCS.     

Fluorescence immunoassay through histone-ds-poly(AT)-templated copper nanoparticles as signal transductors for the sensitive detection of Salmonella choleraesuis in milk

The rapid and sensitive detection of foodborne pathogens is one of the most important issues in food safety control. In this work, we developed a novel fluorescence immunoassay method for the sensitive detection of Salmonella choleraesuis. The method uses the fluorescent signals of histone-ds-poly(AT)-templated copper nanoparticles (His-pAT CuNP) as signal transducers and glucose oxidase as an alternative for horseradish peroxidase for the generation of hydrogen peroxide (H₂O₂) through the catalysis of glucose. The H₂O₂ is then further converted into hydroxyl radical (·OH) by Fenton reagents. Owing to the ultrahigh sensitivity of His-pAT CuNP synthesis toward ·OH, the proposed fluorescence immunoassay method exhibited excellent sensitivity for S. choleraesuis, with a limit of detection of 8.04 × 10¹ cfu/mL, which is 3 orders of magnitude lower than that of the tetramethylbenzidine-based traditional immunoassay. The reliability of the proposed method was evaluated by using spiked milk samples with S. choleraesuis concentration ranging from 8.8 × 10¹ to 8.8 × 10⁴ cfu/mL. The average recoveries for the intra- and inter-assay ranged from 73.52 to 96.59% and from 66.99 to 98.24% with a coefficient of variation from 6.85 to 31.26% and 5.46 to 17.99%, respectively. These results indicated that the proposed fluorescence immunoassay possesses a great potential for ultra-sensitive detection of foodborne pathogens in food safety control.     

Health risks derived from consumption of lettuces irrigated with tertiary effluent containing norovirus

Wastewater is a valuable resource for water-scarce regions, and is becoming increasingly important due to the rising frequency of droughts as a result of climate change. The health risks derived from ingestion of lettuce that has been irrigated with effluent from a wastewater treatment plant (WWTP) in Catalonia (Spain) were estimated following a quantitative microbial risk assessment (QMRA) approach using site-specific data. Norovirus (NoV) was selected for this analysis, since it is the most common cause of acute gastroenteritis outbreaks in Catalonia. Two scenarios, irrigation with secondary and with tertiary effluent, were analysed. An uncertainty analysis was conducted to determine the impact of possible internalization of NoV into edible parts of the lettuce. The mean disease burden for ingestion of lettuce irrigated with secondary and tertiary effluent was 7.8 × 10^− 4 Disability Adjusted Life Years (DALYs) per person per year (pppy) and 3.9 × 10^− 4 DALYs pppy, respectively. A sensitivity analysis revealed that the model parameters with higher influence on the probability of disease are the concentration of NoV in the effluent and the consumption of lettuce. In order to decrease the disease burden to the guidance level of 10^− 6 DALYs pppy, the tertiary treatment should be able to achieve a 4.3 log reduction of the concentration of NoV. If internalization of NoV into lettuces occurs, this would require a reduction of 7.6 log. This is the first time that site specific data and virus internalization in crops are incorporated in a QMRA of irrigation of lettuce and its impact is quantified.     

Sensitive fluorescence ELISA with streptavidin scaffolded DNA tetrads for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7 poses a threat to humans. Traditional ELISA is not a sensitive method for the detection of E. coli O157:H7. Here, an efficient method was designed for improving the load capacity of alkaline phosphatase (ALP) with streptavidin scaffolded DNA tetrad (SS-DNAt). With more ALP, more ascorbic acid 2-phosphate was catalyzed to ascorbic acid that was used to synthesize fluorescence poly adenine-thymine–templated copper nanoclusters. Based on SS-DNAt, fluorescence ELISA was successfully proposed for improving the sensitivity for detection of E. coli O157:H7 in milk samples. The method showed a linear range of 10⁴ to 10⁶ cfu/mL. The limit of detection of fluorescence ELISA was 3.75 × 10³ cfu/mL and 6.16-fold better than that of traditional ELISA. The recovery of the fluorescence ELISA was 86.7 to 93.6% with the coefficient of variation of 5.6 to 10.5% in milk. This method could be used to detect hazardous material in food.     

Sensitive and Selective Determination of Riboflavin in Milk and Soymilk Powder by Multi-walled Carbon Nanotubes and Ionic Liquid [BMPi]PF<Subscript>6</Subscript> Modified Electrode

A novel electrochemical method to detect riboflavin was proposed using a multi-walled carbon nanotubes and ionic liquid N-butyl-N-methyl-piperidinium hexafluorophosphate composite film modified glassy carbon electrode (MWNTs-[BMPi]PF₆/GCE). A well-defined CV behavior with a pair of sensitive and well-shaped redox peak was observed, and the response value of riboflavin at MWNTs-[BMPi]PF₆/GCE is much higher than that at MWNTs/GCE in 0.1 mol L^?1 HAc-NaAc buffer solution (pH 4.5). The electrochemical approach based on a sensitive DPV analytical signal exhibits an excellent analytical performance with a wide linear range (2.6 × 10^?8 to 1.3 × 10^?6 mol L^?1) and low detection limit (4.7 × 10^?9 mol L^?1) for the riboflavin. Moreover, the proposed method possesses a potential practical application value and can be employed for the quantitative analysis of trace riboflavin with a recovery of 95.8–102.4 % in food samples such as milk and soymilk powder.     

Phages of Bacteroides (GB-124): a novel tool for viral waterborne disease control?

Current fecal indicator bacteria (FIB) and emerging microbial source tracking (MST) methods may indicate the presence and even the likely source of water contamination, but they are less effective at determining the potential risk to health from human enteric viruses. This paper investigates the presence of human-specific phages (detected using a low-cost MST method) in municipal wastewaters (MW) and assesses whether they may be used effectively to screen for the likely presence of human adenovirus (HAdV) and norovirus (NoV). The findings demonstrated that all samples positive for HAdV and/or NoV also contained phages infecting Bacteroides GB-124 (mean = 4.36 log(10) PFU/100 mL) and that GB-124 phages, HAdV, and NoV were absent from samples of nonhuman origin. HAdV and NoV were detected more frequently in MW samples containing higher levels of phages (e.g., >10(2)) and FIB (e.g., >10(3)). Interestingly, at one sewage treatment works (STW), the levels of GB-124 phages present in treated MW were not significantly lower (p = 0.001) than those in untreated MW. There was a positive correlation (R = 0.42) between the size of STW and the number of GB-124 phages present in the final treated effluent. Therefore, the detection of GB-124 phages by a simple phage-lysis method may have considerable potential as a low-cost surrogate for the detection of certain human pathogenic viruses in MW and receiving waters.     

Rapid and simple quantitative identification of Listeria monocytogenes in cheese by isothermal sequence exchange amplification based on surface-enhanced Raman spectroscopy

Foodborne pathogens detection is important to ensure food safety and human health. In this study, we designed a comet structure to rapidly and sensitively detect foodborne Listeria monocytogenes. This method combined isothermal sequence exchange amplification (SEA) and surface-enhanced Raman spectroscopy. Listeria monocytogenes DNA could be rapidly amplified at a constant temperature via SEA with a pair of modified primers, which rendered the precise thermal control instrumentation unnecessary. Efficient SEA amplification generated a large number of DNA duplexes that could be easily captured by streptavidin-modified magnetic bead and Au^MB@Ag-isothiocyanate fluorescein antibody (anti-FITC). Au^MB@Ag-anti-FITC was used as a signal probe, which generated a significant excitation signal at 1,616 cm^?1 for quantitative detection and analysis. The results displayed sensitive detection of L. monocytogenes in cheese from 2.0 × 10¹ cfu/mL to 2.0 × 10⁶ cfu/mL within 1.0 h with a detection limit of 7.8 cfu/mL. Furthermore, this comet structure displayed the desirable specificity as its specific primers and amplified DNA ends were attached to streptavidin-modified magnetic beads and Au^MB@Ag-anti-FITC, respectively. We expected that the method devised would provide a promising new approach to screening for L. monocytogenes and guarantee the microbiological safety of dairy products.     

福建省养殖环节牡蛎中诺如病毒污染状况分析

目的 了解福建省养殖环节牡蛎中诺如病毒(Norovirus)污染状况及基因分型,进一步定量分析诺如病毒污染水平,为食品中诺如病毒污染监测及安全风险评估提供数据支持.方法 2017年8月—2018年9月,于福建省某养殖基地采集牡蛎样品共244份,采用实时荧光定量聚合酶链式反应(PCR)法对样品中诺如病毒进行检测,确定其污...     

Development and application of a sensitive,rapid, and reliable immunomagnetic separation-PCR detection method for Cronobacter spp.

Cronobacter spp. have been linked to clinical cases of infection in both adults and infants. Enrichment of Cronobacter spp. before detection has been necessary but is quite time consuming. Hence, we sought to develop an immunomagnetic separation (IMS) PCR method that could shorten the time of enrichment before the detection of Cronobacter spp. The polyclonal antibody used in this immunomagnetic separation was prepared based on the outer membrane protein A of Cronobacter sakazakii China Center of Industrial Culture Collection 21560 and had high specificity to the target. The primers used in the IMS-PCR method also showed high specificity. The detection limit of IMS-PCR for pure C. sakazakii culture was 5.2 × 10² cfu/mL. Cronobacter sakazakii in artificially contaminated powdered infant formula (PIF) was also detected at a detection limit of 5.2 × 10² cfu/mL. After 8 h of enrichment, the detection limit in PIF was lower than 5.2 × 10¹ cfu/mL. An interference test using Escherichia coli in artificially contaminated PIF showed that the IMS-PCR method developed in this study had a good ability to resist interference. Finally, the IMS-PCR method was applied to the detection of Cronobacter in food samples and was shown to be reliable. Thus, this newly developed IMS-PCR detection method was quite sensitive, rapid, and reliable and could be applied to the detection of Cronobacter in foods.     

Multiplex PCR coupled with propidium monoazide for the detection of viable Cronobacter sakazakii,Bacillus cereus,and Salmonella spp. in milk and milk products

Cronobacter sakazakii, Bacillus cereus, and Salmonella spp. are common food-borne pathogens. The aim of this study was to develop a sensitive, specific, and rapid method for the simultaneous detection of these 3 pathogens in milk and milk products. Three specific primers were designed based on ompA, invA, and cesB of C. sakazakii, Salmonella spp. and B. cereus, respectively, for use in a multiplex PCR (mPCR). To eliminate false-positive results, cells were pretreated with propidium monoazide (PMA) for the selective elimination of the genomic DNA of dead cells. An internal amplification control was applied as an indicator of false-negative results from the interference of inhibitors in the food matrix. Results showed that, in pure culture, the limits of detection of the assay for C. sakazakii, Salmonella Enteritidis, and B. cereus were 9.5 × 10⁴, 7.4 × 10², and 7.5 × 10² cfu/mL, respectively. Moreover, 8 cfu/mL of viable B. cereus cells were detected after 5 h of enrichment, and 9 cfu/mL of viable C. sakazakii and 7 cfu/mL of Salmonella Enteritidis were detected after 7 h of enrichment in spiked pure milk, walnut peanut milk, and whole-wheat milk. To validate the PMA-mPCR assay, the PMA-mPCR assay and the traditional culture method were performed to detect the 3 bacterial strains in 1,165 milk product samples. The PMA-mPCR assay obtained the same results as the culture-based method. Results demonstrated that the PMA-mPCR assay has excellent sensitivity and specificity for the simultaneous detection of viable C. sakazakii, Salmonella Enteritidis, and B. cereus in milk and milk products.     

Upconversion Nanoparticles Capped with Molecularly Imprinted Polymer as Fluorescence Probe for the Determination of Ractopamine in Water and Pork

A novel fluorescence probe was designed and synthesized to quantify ractopamine in foods using molecularly imprinted polymer (MIP) as a specific recognition element and YF₃:Yb³⁺, Er³⁺ upconversion nanoparticles (UCNPs) as a fluorescence signaling component. The developed UCNP@MIP probe was characterized using X-ray powder diffraction, scanning electron microscopy, and thermogravimetric analysis. The fluorescence of UCNP@MIP probe was quenched specifically by ractopamine, and good linear regression ranging from 1.66 × 10^?8 to 3.3 × 10^?7 mol/L was obtained with the limit of detection of 8.6 × 10^?10 mol/L. This method was further applied to determine ractopamine in water and pork samples. Recoveries between 76.61 and 88.97% were obtained with relative standard deviation ranging from 1.15 to 2.67% (n = 3). This UCNP@MIP probe combined high selectivity of MIP and high sensitivity of upconversion fluorophore and showed potential to determine various food chemical hazards.