Intra-laboratory validation of a concentration method adapted for the enumeration of infectious F-specific RNA coliphage, enterovirus, and hepatitis A virus from inoculated leaves of salad vegetables

Salad vegetables exposed to fecal contamination may cause outbreaks of hepatitis or gastro-enteritis if they are eaten raw. A procedure, based on elution with phosphate-buffered saline and concentration by filtration through membrane filters, was developed for the recovery of enteric viruses from salad leaves. The method was evaluated using lettuce leaves inoculated with hepatitis A virus (HAV), poliovirus, and MS2 bacteriophage. In addition, this method was validated by an intra-laboratory study using leaves of various salad vegetables inoculated with MS2 phage. The French standard NF V 03-110 was used to establish the general principle and the technical protocol of the validation procedure. Linear regression models describing the quantitative reactions were good fits to data in the whole range of viral concentrations tested, which was from about 1 to 4 log plaque-forming units (PFU) per 25 g of lettuce. The fractions of inoculated viruses recovered were estimated to be about 64% for HAV, 18% for poliovirus, and 29% for MS2. No significant effect of the food matrix was found using various types of salad vegetable (butter lettuce, iceberg lettuce, romaine lettuce, witloof chicory, curly endive, corn salad, rocket and watercress). Moreover, the variance of the results was constant for all levels of virus contamination within the experimental range. Intermediate reproducibility experiments were also performed to allow calculation of the uncertainty factor, which was found to be 0.58 log PFU/25 g. When used in association with phage enumeration, this validated procedure is rapid enough to be used for screening salad vegetables for evaluation of the efficacy of processes for control of pathogenic microorganisms on such foods.     

An ultracentrifugation-based approach to the detection of hepatitis A virus in soft fruits

A method was developed for detection of hepatitis A virus (HAV) in soft fruits (raspberries and strawberries). After washing the sample in 1 M sodium bicarbonate with added soya protein, fruits were removed by slow speed centrifugation, then particulate material and residual pectin were removed from the supernatant by flocculation and pectinase treatment during another slow speed centrifugation. Virus particles were then sedimented by ultracentrifugation. RNA was extracted from the virus particles, and nested RTPCR was performed on the nucleic acid extract. Nested RTPCR comprised an RTPCR, followed by PCR to amplify sequences within the amplicon. Internal amplification controls (IACs) were constructed for both the RTPCR and the PCR. The sensitivity of the nested RTPCR was approximately 10 RTPCRU. The overall method was shown to be able to detect 10(4) RTPCRU HAV in 90 g fresh strawberries, and 10(3) RTPCRU HAV in 60 g fresh raspberries. It is estimated that the lowest possible limit of detection of the method should be between 40 and 400 RTPCRU HAV per fruit sample. The method can be performed within one day, in suitably equipped microbiological laboratories, and is suitable for routine screening of food samples, and for analysis of suspected samples, e.g. during outbreak investigations.     

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.     

Anti-inflammatory effects of phytochemicals from fruits,vegetables, and food legumes: A review

Inflammation is the first biological response of the immune system to infection, injury or irritation. Evidence suggests that the anti-inflammatory effect is mediated through the regulation of various inflammatory cytokines, such as nitric oxide, interleukins, tumor necrosis factor alpha-α, interferon gamma-γ as well as noncytokine mediator, prostaglandin E₂. Fruits, vegetables, and food legumes contain high levels of phytochemicals that show anti-inflammatory effect, but their mechanisms of actions have not been completely identified. The aim of this paper was to summarize the recent investigations and findings regarding in vitro and animal model studies on the anti-inflammatory effects of fruits, vegetables, and food legumes. Specific cytokines released for specific type of physiological event might shed some light on the specific use of each source of phytochemicals that can benefit to counter the inflammatory response. As natural modulators of proinflammatory gene expressions, phytochemical from fruits, vegetables, and food legumes could be incorporated into novel bioactive anti-inflammatory formulations of various nutraceuticals and pharmaceuticals. Finally, these phytochemicals are discussed as the natural promotion strategy for the improvement of human health status. The phenolics and triterpenoids in fruits and vegetables showed higher anti-inflammatory activity than other compounds. In food legumes, lectins and peptides had anti-inflammatory activity in most cases. However, there are lack of human study data on the anti-inflammatory activity of phytochemicals from fruits, vegetables, and food legumes.     

A robust GC-MS/MS method for the determination of chlorothalonil in fruits and vegetables

Chlorothalonil is a non-systemic fungicide that is easily degraded in contact with plants and soil or even by the effect of light and pH. A method for the determination of chlorothalonil in courgettes, strawberries, oranges, leeks and tomato by solvent extraction followed by GC-MS/MS with a triple quadrupole analyser was developed. The causes of chlorothalonil degradation during sample treatment were studied and minimised. The final method was based on extraction with acetone in the presence of 0.1?M EDTA sodium salt solution, and clean-up by SPE using OASIS HLB cartridges. Isotope-labelled hexachlorobenzene (HCB-¹³C₆) was added as an internal standard to the SPE extracts before analysis by GC-MS/MS (EI) (QqQ) analysis in order to correct for instrumental deviations. Quantification was performed by matrix-matched standard calibration using relative responses to the internal standard. Two MS/MS transitions were used for mass spectrometric determination of chlorothalonil to ensure reliable quantification and confirmation. The method was validated using blank samples (for all matrices) spiked at two levels. Recoveries between 77% and 110% and an RSD below 20% were obtained for 0.1 and 0.01?mg?kg^?1 spiking levels (n?=?5). The validated method was applied to treated and untreated samples collected from an experimental field where a chlorothalonil formulated was applied.     

A non-perturbing scheme for the mineralogical characterization and quantification of inorganic colloids in natural waters

Although the role played by inorganic colloids in natural waters depends on their composition as well as on their size, the characterization of submicron particles has rarely gone beyond describing the morphology and identifying some of the most abundant particles. The process of quantification has been hampered by a lack of suitable analytical methods. This study demonstrates that it is possible to identify and quantify inorganic particles in the colloidal size range by applying a straightforward methodology based on a well-proved, quantitative, and nonperturbing method of sample preparation (direct centrifugation of the samples on transmission electron microscopy grids) in conjunction with particle analysis using widely available techniques: transmission electron microscopy, energy dispersive X-ray spectroscopy (EDS), and selected area electron diffraction (SAED). The method has successfully been applied to six water samples from basins of contrasting geological characteristics. The method has the advantage of minimizing sample modifications by allowing on site sample preparation, using standard equipment, and it is not particularly time-consuming. Notably, the combination of EDS and SAED information makes it possible to characterize and quantify the most abundant components of the colloidal pool in the majority of the aquatic systems: the different types of aluminosilicates.     

自然冷源在果蔬贮藏保鲜中的应用效果研究

利用一套自制的新式制冰系统,以芹菜和草莓为材料,验证利用自然冷源贮藏果蔬的效果。结果表明:贮藏温度均为1.1℃的条件下,普通机械冷库相对湿度为72%,自然冷源冷库相对湿度则保持在88%;贮藏芹菜41d时前者失重率为62.47%,后者23.63%;贮藏草莓21d时,前者失重率为15.76%,后者只有6.15%,且后者在降低腐烂率,减少糖度、色泽、Vc损失方面效果明显。     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.     

Recent developments in high efficient freeze-drying of fruits and vegetables assisted by microwave: A review

Microwave heating has been applied in the drying of high-value solids as it affords a number of advantages, including shorter drying time and better product quality. Freeze-drying at cryogenic temperature and extremely low pressure provides the advantage of high product quality, but at very high capital and operating costs due partly to very long drying time. Freeze-drying coupled with a microwave heat source speeds up the drying rate and yields good quality products provided the operating unit is designed and operated to achieve the potential for an absence of hot spot developments. This review is a survey of recent developments in the modeling and experimental results on microwave-assisted freeze-drying (MFD) over the past decade. Owing to the high costs involved, so far all applications are limited to small-scale operations for the drying of high-value foods such as fruits and vegetables. In order to promote industrial-scale applications for a broader range of products further research and development efforts are needed to offset the current limitations of the process. The needs and opportunities for future research and developments are outlined.     

A real-time PCR method for the detection and quantification of lupin flour in wheat flour-based matrices

Lupin flour is growingly being used in bakery products, mainly as a soybean protein substitute. The aim of the present work was to detect and quantify the presence of lupin flour in wheat-based foods using a newly set up qPCR system based on SYBR green. Although DNA sequence information for lupin is scarce, it has been possible to design a primer pair highly specific for the target gene and devoid of any primer-dimers amplification capacity. Lupin flour revealed to be a difficult matrix, since large amounts of compounds tend to co-purify with DNA, even adopting well established extraction protocols. Nonetheless, the primers used allowed to reach high PCR efficiencies and did not show any cross-reactivity with DNAs extracted from various plant and animal foods. The sensitivity achieved was 7 pg of lupin DNA, corresponding to a percentage of less than 0.1% of lupin flour in the foods.     

Multiplex real-time PCR for the detection and quantification of DNA from beef, pork, chicken and turkey

Many meat products are composed of two or more meat species. To determine the proportion of these meat fractions, a quantitative multiplex PCR was developed for the quantification of beef, pork, chicken and turkey. This system proved its applicability, precision and accuracy in examining different meat products from the market. Thus it allows the efficient control of composed meat products in official food control and production control laboratories.     

Multiplex real-time PCR for the detection and quantification of DNA from beef,pork, horse and sheep

Meat products are often composed of more than one meat species. A quantitative multiplex PCR was developed to determine the proportion of meat fractions of beef, pork, horse and sheep. The precision and accuracy were investigated by dilutions of DNA from all four species and examining different meat products from the market. Application of this tetraplex quantitative real-time PCR system will enable official food control and production control laboratories to efficiently investigate the composition of meat products.     

Detection of human norovirus in cherry tomatoes, blueberries and vegetable salad by using a receptor-binding capture and magnetic sequestration (RBCMS) method

In this study, we developed a sensitive receptor-binding capture and magnetic sequestration (RBCMS) method capable of concentrating human norovirus (HuNoV) from various food samples within few hours. We found that distilled water was suitable for the elution of HuNoV from inoculated tomatoes and blueberries, and glycine buffer improved the elution of HuNoV from inoculated salad. A significant improvement in post-extraction RNA yield was achieved by sequentially heat-releasing and column-extracting over either technique alone. The viral recovery of the RBCMS method was significantly higher than both the same-day PEG method (90 min PEG precipitation) and the two-day PEG method (overnight PEG precipitation) with a recovery rate of 8.75%, 1.03% and 5.40%, respectively. The detection limit of HuNoV by RBCMS method was significantly improved to 0.056 RTU. The estimated minimal concentration powers (MCPs) were 6.11, 30.48, and 63.60-fold for the same-day PEG, two-day PEG, and RBCMS methods, respectively. RNase protection assay suggests that the viral genome was protected from RNase attack by remaining within the viral capsid. The signal detected by the RBCMS method might be more biologically relevant, as it requires both intact viral capsid to bind to HBGA receptors and the presence of viral genome to be amplified. Overall, the RBCMS method takes significantly less time than current PEG precipitation methods, recovers a higher yield of HuNoV from various food samples, and hence exhibits higher sensitivity.     

Multiplex real-time PCR for the detection and quantification of DNA from duck, goose, chicken, turkey and pork

Meat products made from liver of poultry like duck and goose are popular and often sold as specialities for high prices. As the prices for the basic raw material are high, fraud may be attractive for producers. To prevent consumers from fraud, official control authorities survey such products. In this work, a quantitative multiplex PCR was developed determining the proportion of DNA and meat fractions of turkey, chicken, duck, goose and pork. The precision and accuracy of the PCR system was investigated. To examine the possibility of determining the meat fractions according to the recipe, reference material was produced and different liver–meat products from the market were analysed. For major components, the measurement uncertainty revealed to be at 39 %. For minor components, it was estimated to be 124 %. The results showed that this pentaplex real-time-PCR system is suitable to control the meat properties of such products although measurement uncertainty may be high.     

Detection of the frequency, antimicrobial susceptibility, and genotypic discrimination of Aeromonas strains isolated from municipally treated tap water samples by cultivation and AP-PCR

The frequency, antibiotic susceptibility, and genotypic discrimination of Aeromonas strains isolated from municipally treated drinking tap water distribution systems were investigated in this study. We have analyzed 148 tap water samples collected from 8 different locations by bacterial cultivation and arbitrarily primed polymerase chain reaction (AP-PCR). Gram negative, hemolytic, oxidase (+) and catalase (+) bacterial colonies were applied to the study. Identification of bacterial colonies was done by conventional biochemical method and API ID 20E panel (BioMerieux-France). Molecular epidemiological discrimination of the isolates was done by AP-PCR. Aeromonas spp. was detected in 6 of 148 (4%) tap water samples from 8 different locations. Five isolates were identified as Aeromonas hydrophila and one isolate was identified as Vibrio fluvialis by conventional biochemical method. These data were also confirmed by API 20E panel. One of 6 isolates was resistant to gentamicin, 2 of 6 isolates were resistant to trimethoprim/sulfamethoxazole, 4 of 6 isolates were resistant to ampicillin and ampicillin-sulbactam and all of 6 isolates were resistant to cephalothin. All isolates were found to be susceptible to amikacin, aztreonam, ceftazidime, ceftriaxone, ciprofloxacin. All 6 strains of Aeromonas were discriminated by AP-PCR and were determined that all isolates were from different genotypic sources. Although the frequency of the isolates was under the standard limits, the results indicate that hemolytic A. hydrophila are present in municipally treated tap water samples in Mersin City. While all strains were genotypically distinct, all of them were resistant to first generation beta lactam antibiotics tested in this study.     

A rapid, sensitive and economic method for the detection, quantification and confirmation of aflatoxins

Summary A rapid, sensitive and economic method for the detection, quantification and confirmation of aflatoxins is described. Aflatoxins B₁, B₂, G₁, and G₂, are extracted by methanol/water (85+15) and partitioned into methylene dichloride. The methylne dichloride solution is cleaned up on a polypropylene column, filled with 0.5 g silica gel 60. The aflatoxins are eluted with cloroform-acetone (90:10) and are detected using bidirectional thin-layer chromatography (TLC) with aluminium silica gel foil. The mean recovery of aflatoxins B₁, B₂, G₁, and G₂, in corn samples was 73, 78, 80, and 64%, respectively; the limit of detection was 0.5 g/kg. The results can also be confirmed by derivative formation using trifluoroacetic acid on the TLC plate. The method has been applied to a wide range of foods with good results.

---

Eine schnelle, empfindliche und kostengünstige Methode zum Nachweis, zur Bestimmung und Bestätigung von Aflatoxinen

Zusammenfassung Es wird eine schnelle, empfindliche und kostengünstige Methode zum Nachweis, Bestimmung und Bestätigung von Aflatoxinen beschrieben. Die Aflatoxine B₁, B₂, G₁ und G₂ werden mit Methanol/Wasser (85+15) extrahiert und in Dichlormethan überführt. Der Dichlormethanextrakt wird auf einer mit 0,5 g Kieselgel 60 gefüllten Polypropylensäule gereinigt. Die Aflatoxine werden mit Chloroform/Aceton (90+10) eluiert und mit zweidimensionaler DC auf Kieselgel-Alufolien nachgewiesen. Die mittleren Wiederfmdungsraten für die Aflatoxine B₁, B₂, G₁ und G₂ in Maismehl betragen 73, 78, 80 und 64%, die Nachweisgrenzen liegen durchschnittlich bei 0,5 g/kg. Zur Bestätigung verdächtiger Befunde kann auf der Platte mit Trifluoressigsäure derivatisiert werden. Die Methode ist bisher an einer Vielzahl von verschiedenen Lebensmitteln mit gutem Erfolg getestet worden.