RT-PCR法检测贝类中的甲肝病毒的研究

在世界范围内,甲肝病毒是与食用贝类有关的主要传染性疾病之一。由于贝类中含有PCR抑制剂以及病毒富集过程中病毒的回收率低,阻碍了天然污染的贝类中HAV的PCR检测。研究中建立了一种经苷氨酸缓冲液洗涤,2次PEG沉降富集病毒,然后进行RNA提取和RT-PCR对贝类中的甲肝病毒进行检测的方法。经比较,采用小体系肠道样品检测比采用全贝检测的富集效果更佳,并比较了PEG8000和PEG6000对病毒富集的效果,回收率分别为13.5%和7.6%,此方法可有效地降低PCR抑制剂的影响,最低检测限可达10个TCID50/1.5 g。     

Norovirus, hepatitis A virus and enterovirus presence in shellfish from high quality harvesting areas in Portugal

This is the first report on the screening of shellfish from Portugal for the presence of human enteropathogenic viruses. Approximately 2000 shellfish (Curbicula fluminea, Ruditapes decussatus, Tellina crassa, Spisula solida, Dosinia exoleta, Ensis spp., Mytilus spp., Ostrea edulis and Cerastoderma edule), organized in 49 batches, were collected between March 2008 and February 2009. They were tested for norovirus (NoV), hepatitis A virus (HAV) and enterovirus (EV) by RT-PCR followed by nucleotide sequencing. Bacterial contamination was also evaluated by Escherichia coli counts. Viral contamination was detected throughout the year in all shellfish species and in all collection areas, independently of their harvesting areas classification. Overall, 67% of all analyzed batches were contaminated by at least one of the studied viruses while the simultaneous presence of two and three viruses was detected in 22% and 6% batches, respectively. Of the three viruses, NoV was detected in 37% of the batches, followed by EV in 35%, and HAV in 33%. Nucleotide sequencing of the NoV and HAV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.4 and HAV subgenotype 1B. The presence of NoV and HAV in shellfish from “A class” harvesting areas of Portugal can represent a potential health risk.     

Rapid and Sensitive Detection of Staphylococcus aureus in Food Using Selective Enrichment and Real-Time PCR Targeting a New Gene Marker

Staphylococcus aureus is a bacterial pathogen considered a principal etiological agent of food poisoning. The aim of this study was to develop and evaluate a rapid and sensitive method for the detection of S. aureus in food by using selective enrichment and a new species-specific real-time polymerase chain reaction (PCR). Specific primers and a TaqMan probe targeted to specific S. aureus gene encoding for acriflavine resistance protein were designed. The real-time PCR was highly specific for S. aureus with 100% inclusivity and 100% exclusivity determined using 83 S. aureus strains and 64 non-S.-aureus strains. PCR detection limit of 6.8 × 10¹ and 3.4 × 10¹ CFU ml⁻¹ were obtained with 100% and 70% detection probability, respectively. The single selective enrichment based on the study of different enrichment conditions was selected and a lysis by boiling was used to obtain bacterial DNA. Out of 112 food samples analyzed, 61 were positive by the PCR-based method and 53 by the standard method. Out of ten food matrices artificially contaminated at a level of 10° CFU g⁻¹, ten and six were positive by the respective methods. Moreover, 10° CFU 10 g⁻¹ was detected in all ten artificially contaminated samples after a large-scale enrichment using PCR-based detection, in contrast to seven false negative by standard detection. The developed method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to microbiological method with the potential for providing of improved food-processing hygiene control.     

Rapid and Sensitive Determination of L-carnitine and Acetyl-L-carnitine in Liquid Milk Samples with Capillary Zone Electrophoresis Using Indirect UV Detection

The purpose of this study was to establish a new capillary zone electrophoresis method with indirect UV detection for determining the concentration of L-carnitine and acetyl-L-carnitine in liquid milk samples. Orthogonal experimental design was applied to arrange and optimize the experimental conditions. Under selected conditions (using melamine as the additive), both L-carnitine and acetyl-L-carnitine could be detected in 6 min, with satisfied reproducibilities (RSD% of migration times <0.8%), linearities (r ² > 0.995), recoveries (>90%), and relative low LODs (3.0 and 5.0 μmol/L for L-carnitine and acetyl-L-carnitine, respectively). The proposed method was successfully applied to the assay of nine brands of bovine milk samples and shows potential for analyzing L-carnitine and acetyl-L-carnitine in other biosamples.     

Analysis of Porcine Gelatin DNA in a Commercial Capsule Shell Using Real-Time Polymerase Chain Reaction for Halal Authentication

The presence of pig derivatives, such as porcine gelatin, in any products is prohibited to be consumed by Muslim community. This study is intended to develop a specific primer from mytochondrial D-loop capable of amplifying DNA from porcine gelatin in commercial capsule shells. Two pairs of primers designed from mitochondrial D-loop region were tested in order to confirm the primer specificity in gelatin sources (pork, beef, and catfish) and fresh tissue (pig, cows, goat, chickens, and rat). Primers were then used to perform sensitivity test of six dilution series (1000, 200, 100, 10, 5, and 1 pg/µL) of porcine gelatin and porcine capsule shell. The amplification was also performed on capsule shell from porcine-bovine mixture gelatin at 0, 10, 20, 30, 40, 50, and 100% concentration. The repeatability test was performed by measuring amplification capsule shells from porcine–bovine gelatin mixture. Real time polymerase chain reaction method using primers designed was further applied to analyze capsule shells purchased from markets. From two primers have been designed specifically, only primer D-Loop 108 (forward: 5’-CGT ATG CAA AAA ACC ACG CCA-3’; reverse: 5’-CTT ACT ATA GGG AGC TGC ATG-3’) had the capability to identify the presence of porcine DNA in fresh tissue and gelatin sources at optimum annealing temperature of 58.4ºC. Sensitivity of the developed method expressed as limit of detection of DNA in gelatin and capsule shells is 5 pg.     

烟草PVY Real-Time PCR定量检测体系的建立及应用

马铃薯Y病毒是近年来危害烟草生产的重要病毒之一,严重影响烟草的产量与品质。本研究利用DNAMAN软件对GenBank数据库中已登录的马铃薯Y病毒(Potato virus Y,PVY)全基因组序列进行序列比对,设计引物,以烟草肌动蛋白基因为内参,建立了烟草PVY的实时定量检测体系。获得的real-time PCR扩增基线平整,指数扩增明显,斜率大;稳定性和重现性好,变异系数小;循环阈值与PCR起始模板量对数之间存在良好的线性关系。与DAS-ELISA相比,该方法具有高效、灵敏、特异性强等优点,为从分子生物学水平上检测烟草中PVY提供了新的技术手段。     

Simultaneous Detection and Differentiation of Campylobacter jejuni, C. coli, and C. lari in Chickens Using a Multiplex Real-Time PCR Assay

A multiplex real-time PCR (qPCR) assay was developed for simultaneous detection and differentiation of the three most important Campylobacter species in chickens. Three novel sets of PCR primers and TaqMan probes were designed to amplify the unique DNA sequences within the hipO, cdtA, and pepT genes which are specific to Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, respectively. To avoid competition in the multiple target amplifications, the concentrations of primers and probes were optimized. By using the optimized qPCR conditions together with a minor-groove binding probe of pepT, amplification efficiency greater than 92% and detection sensitivity of 38 genome copies/reaction have been achieved for all three targets. The assay was highly specific for C. jejuni, C. coli, and C. lari with testing of 33 Campylobacter strains and 20 non-Campylobacter strains. In chicken samples spiked with known quantities of Campylobacter cells, the assay was able to detect 1 CFU/g after a 24-h enrichment. Application of the assay in food was further evaluated using 21 fresh chicken samples obtained from local supermarkets. The results revealed that, after a 24-h or 48-h enrichment, 14 samples (66.7%) were positive for C. jejuni, five samples (23.8%) were positive for C. coli, and none of the samples was contaminated by C. lari. Taken together, the multiplex qPCR assay combined with an enrichment step is a sensitive, species-specific, and non-labor-intensive method suitable for rapid detection of C. jejuni, C. coli, and C. lari in chicken samples.     

Detection and characterization of pathogenic vibrios in shellfish by a Ligation Detection Reaction-Universal Array approach

Vibrios are a group of major foodborne pathogens widely distributed in marine environment. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health. However, other vibrios, e.g. Vibrio alginolyticus, Vibrio mimicus and Grimontia hollisae, apparently less relevant in the group of foodborne pathogens, have been sporadically found in outbreaks. For seafood safety and economic purposes, a rapid and powerful method for the specific identification of harmful Vibrio strains is needed. We developed a PCR-Ligase Detection Reaction-Universal Array (PCR-LDR-UA) assay for the simultaneous identification of pathogenic vibrios and detection of virulence coding genes. The entire procedure was validated on a total of 31 reference strains and isolates from clinical and environmental samples, as well as on bivalve tissue homogenates infected with different strains of target Vibrio species. Twenty-three shellfish samples directed to human consumption were successfully screened, thus demonstrating that the developed microarray-based platform could be a reliable and sensitive detection tool for the identification of harmful Vibrio strains in seafood.     

In-house Validation of a DNA Extraction Protocol from Honey and Bee Pollen and Analysis in Fast Real-Time PCR of Commercial Honey Samples Using a Knowledge-Based Approach

Food Analytical Methods - For consumers, honey is a natural product that should not be subjected to treatment or alteration. Since the question of the presence of genetically modified organisms...     

Polymerase Chain Reaction for the Rapid Detection and Serovar Identification of Salmonella in Food and Feeding Stuff

The polymerase chain reaction (PCR) is one of the most important rapid methods for the sensitive and specific detection of pathogenic and spoilage microorganisms. The method is increasingly applied in surveillance and monitoring programs to detect pathogens, especially for ensuring the safety and quality of food. The food-borne pathogen Salmonella together with Campylobacter is the most predominant bacterial pathogen in Europe, causing approximately 160,000 confirmed human cases per year. During the last two decades, the importance of Salmonella for food safety triggered the development of dozens of PCR-based detection methods. They promise significant advantages compared to the traditional culture-based methods with respect to speed, easiness, reliability, sensitivity, cost effectiveness, and automation. However, many of them are not applicable because of lacking validation procedures. Meanwhile, PCR has internationally been standardized and guidelines as well as standards for the validation of alternative methods, such as PCR, were established. This review will give an overview of the historical development of PCR-based methods for the detection of Salmonella including validation aspects and summarizes the state-of-the-art for the detection of Salmonella in food and feeding stuff by real-time PCR. Furthermore, current multiplex PCR-based serovar-specific identification methods will be reviewed.     

Sensitive, Rapid and Precise Determination of L-Glutamic Acid in Cheese using a Flow-Injection System with Immobilized Enzyme Column

Glutamic acid in cheese was determined by its reaction in a packed-bed enzyme reactor containing immobilized glutamate dehydrogenase (GIDH) in a flow-injection system, measuring fluorescence of NADH generated. There was a linear relationship (r = 0.999) between log-peak height and log-glumatic acid concentration (0.01 to 0.5 mM). The detection limit was 0.005 mM for an injection volume of 88 μX. The sample frequency was 30/h and the precision was better than 1.2% for 10 successive assays. The accuracy of the enzyme reactor-flow injection system was evaluated by comparison with HPLC results and an excellent correlation was obtained (r = 0.996). No measurable decrease in activity of the GIDH column was observed after a 3-month period.     

Rapid and Non-Destructive Detection of Decay in Peach Fruit at the Cold Environment Using a Self-Developed Handheld Electronic-Nose System

Non-destructive detection of decay in fruit in real time during cold chain is important to recycle the decayed fruit in time for reverse supply chain. Fruit is commonly stored inside external packages during cold chain, making neither manual observation nor optical inspection techniques available to detect the decay in fruit. In this work, the potential of a self-developed handheld electronic nose (e-nose) instrument to non-destructively acquire volatile substances and then detect decay in peach fruit during cold chain (0 °C) was explored. A desktop e-nose instrument was considered as a comparison. The storage days of peach fruit during storage were also predicted by two instruments. Partial least squares discriminant analysis and least squares support vector machines (LS-SVM) were used for the classification of decay in peach fruit. Partial least squares regression and LS-SVM were used for the prediction of the storage days. Successive projection algorithm (SPA), uninformation variable elimination (UVE), UVE-SPA, and competitive adaptive reweighted sampling were applied to select the characteristic variables from e-nose data. The best model for the classification of decayed fruit during cold chain by the handheld e-nose instrument had the correct answer rate of prediction of 95.83% (94.64% for healthy samples and 100.00% for decayed samples). The best model for predicting the storage days of peach fruit during cold chain by the handheld e-nose instrument had the residual predictive deviation value of 9.283. The results indicate that the self-developed handheld e-nose system is a simple and non-destructive tool to detect decay in peach fruit during cold storage.     

Development of Sensitive,Rapid, and Effective Immunoassays for the Detection of Vitamin B<Subscript>12</Subscript> in Fortified Food and Nutritional Supplements

An indirect competitive enzyme-linked immunosorbent assay (Ic-ELISA) method and lateral-flow immunochromatographic (ICA) strip assay method were developed for the detection of vitamin B₁₂ (four major forms, cyanocobalamin, hydroxocobalamin, adenosylcobalamin, and methylcobalamin) in different food products. The limit of detection and 50 % inhibitory concentration for the Ic-ELISA method were 0.065 and 0.43 ng/mL, respectively. The visual limit of detection and cutoff value for the lateral-flow ICA strip assay method were 1 and 4 ng/mL, respectively. For the detection of fortified food and nutritional supplements in vitamin tablets, energy drink, and infant milk powder samples, the recovery rates were in the range of 81 to 122 % for the Ic-ELISA method, and even the lowest content in infant milk powder was identified by the lateral-flow ICA strip assay. Therefore, both of these methods are sensitive, rapid, and effective and are suitable for the on-site detection and rapid screening of mass samples.     

Rapid Detection of Chemical Hazards (Toxins,Dioxins, and PCBs) in Seafood

Among the various hazards occurring in fish and seafood chemical hazards and in particular toxins (ciguatera, scombroid fish poisoning, paralytic shellfish poisoning, neurotoxic (brevetoxic) shellfish poisoning, puffer fish poisoning, diarrhetic shellfish poisoning) have an important place in food poisoning cases. On the other hand, some of the chemical hazards are often due to the pollution of the environment (heavy metals, dioxins, polychlorinated biphenyls, and halogenated aromatic hydrocarbons) and their detection is neither rapid nor facile. As a result there was a great need for developing new rapid and effective methods toward the chemical hazards determination mainly because of their high toxicity. The aim of this review is to provide the information about the new up-to-date detection techniques (Immunological, Chemical and Biochemical, and Molecular assays) in conjunction with detection limits. The latter is made possible by means of inclusion of seven comprehensive and, in most case cases, very extended tables. A reference is also made on the risk characterization of toxins as regards their importance to food contamination or poisoning.     

Detection and Rapid Purification of Internalin B as a Protein Marker in Listeria monocytogenes

Clinical and food strains of Listeria monocytogenes have been found to express InternalinB (InlB) without polymorphism. InlB, which is a 67-kDa surface protein, behaves as an invasion and adhesion protein of the bacterium into cells. Thus, InlB could be a good candidate as a protein marker to detect L. monocytogenes. Three strains of L. monocytogenes (ATCC 19115, serotype 4b, ATCC 19111, serotype 1/2a, ATCC 7644, serotype 1/2c) were tested to detect and purify InlB. L. grayii (ATCC 25400) and L. innocua (isolated from soft cheese) were used as controls that did not express InlB due to the absence of its gene on the chromosome. InlB was quantitatively extracted in a solubilized form by treatment of L. monocytogenes with 1 M Tris-Cl at pH 7.5. Immunoblot analysis using anti-InlB polyclonal antibody revealed that L. monocytogenes 19115 had the lowest expression, which required enrichment of InlB for its detection. Simple spin-ion exchange chromatography with strong acidic cation exchanger was used to enrich the InlB protein that is strongly basic. Most impurities in the column were washed with 25 mM sodium acetate whereas the InlB protein was only protein retained in the column and can be eluted by 1 M NaCl. The data presented here showed that spin-ion exchange chromatography was found to be a simple and rapid method to enrich and purify InlB within 20 min.     

Comparison of Rapid Methods for Detection of Giardia spp. and Cryptosporidium spp. (Oo)cysts Using Transportable Instrumentation in a Field Deployment

Reliable, sensitive, quantitative, and mobile rapid screening methods for pathogenic organisms are not yet readily available, but would provide a great benefit to humanitarian intervention units in disaster situations. We compared three different methods (immunofluorescent microscopy, IFM; flow cytometry, FCM; polymerase chain reaction, PCR) for the rapid and quantitative detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in a field campaign. For this we deployed our mobile instrumentation and sampled canal water and vegetables during a 2 week field study in Thailand. For purification and concentrations of (oo)cysts, we used filtration and immunomagnetic separation. We were able to detect considerably high oo(cysts) concentrations (ranges: 15-855 and 0-240 oo(cysts)/liter for Giardia and Cryptosporidium, respectively) in 85 to 300 min, with FCM being fastest, followed by PCR, and IFM being slowest due to the long analysis time per sample. FCM and IFM performed consistently well, whereas PCR reactions often failed. The recovery, established by FCM, was around 30% for Giardia and 13% for Cryptosporidium (oo)cysts. It was possible to track (oo)cysts from the wastewater further downstream to irrigation waters and confirm contamination of salads and water vegetables. We believe that rapid detection, in particular FCM-based methods, can substantially help in disaster management and outbreak prevention.     

Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes

Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx ₁, stx ₂, and wzy _O157 genes in combination with probes and primers targeting either the fliC _h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx ₁, stx ₂, wzy _O157, and eae genes; however, using the assay targeting the stx ₁, stx ₂, wzy _O157, and fliC _h7 gene combination, a positive result was always obtained for the fliC _h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC _h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx ₁, stx ₂, eae, and wzy _O157 or the fliC _h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC _h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.     

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.