Development of triplex PCR for simultaneous detection of maize,wheat and soybean

A reliable and fast detection of important food plants, such as maize (Zea mays L.), wheat (Triticum aestivum L.), and soybean (Glycine max L.) is of particular interest for food authenticity and safety assessment. In this study, the novel multiplex polymerase chain reaction (PCR) method was developed for the rapid qualitative detection of soybean, maize and wheat. To this purpose, new soybean-specific and maize-specific PCR primers were designed. Their specificity was assayed by uniplex PCRs with different plant species, namely maize, soybean, wheat, oats (Avena sativa), and barley (Hordeum vulgare L). Gel electrophoresis of the amplification products demonstrated high specificity of both primer pairs for identification of relevant species. Subsequently, based on the developed DNA markers, the species-specific triplex PCR targeting maize invertase gene, soybean lectin gene and wheat low-molecular-weight glutenin subunit was developed and optimized for simultaneous identification of these three plant species. The developed PCR method enables specific, effective and rapid detection of maize, wheat and soybean and may be used for food analysis.     

Development and evaluation of a real-time PCR assay for detection of lobster,a crustacean shellfish allergen

Crustacean shellfish are a leading cause of food allergy in American adults, and the Food Allergen Labeling and Consumer Protection Act requires that different types of crustacean shellfish be distinguished from each other. In general ELISA assays are not capable of differentiating crustacean type, but PCR assays are. In this work, a real-time PCR assay for lobster, a crustacean shellfish allergen, was developed and evaluated. Food matrices were spiked with lobster meat at 0.1, 1, 10, 100, 1000, 10⁴, and 10⁵ parts per million (ppm). In addition to testing of several different food matrices, method performance was determined using conditions which have historically proven challenging for PCR analyses, specifically food matrices with low DNA content and acidic pH levels, as well as foods that were treated with combined high temperature and pressure. Real-time PCR standard curves were generated from spiked, treated foods and analyzed with respect to linear range and reaction efficiency. In most cases, the assay was linear over 6–8 orders of magnitude; lower limits of detection were 0.1–1 ppm and reaction efficiencies were within the preferred range of 100 ± 10%. A notable exception occurred in the case of heat treatment at acidic pH, which resulted in severe delay or complete loss of amplification signals.     

Fungal and aflatoxin contamination of marketed spices

Fourteen spice samples were collected from local markets in Doha, Qatar, during 2012, and were surveyed for the presence of potentially harmful mycoflora and for contamination with aflatoxins B₁, B₂, G₁, and G₂ by high-performance liquid chromatography (HPLC). Among the tested spice samples, chili powder showed the highest presence of fungal propagules, while ginger, curry and garlic samples did not present any fungal contamination. A total of 120 isolates, mostly belonging to Aspergillus and Penicillium genera, were collected and 33 representative species were identified by amplification and sequencing of the internal transcribed spacer (ITS) region. Aspergillus flavus, Aspergillus nomius and Aspergillus niger were the most dominant. Thirty-seven Aspergillus strains were screened for their potential to produce aflatoxins using biochemical and molecular tools: only 9 A. flavus strains showed both fluorescence and amplification with all the three primers targeting aflP, aflM and aflR genes. Aflatoxins were detected in five spices (black pepper, chili, tandoori masala. turmeric and garam masala), and with the exception of garam masala, the tested samples of turmeric, black pepper, tandoori masala and chili powder exceeded B₁ and/or total aflatoxin maximum levels. Our results demonstrate the potential for mycotoxin biosynthesis by fungi contaminating imported spice products.     

Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus,Listeria monocytogenes and Salmonella spp. in raw shrimp

Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efficient multiplex real-time PCR for the simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp without a prior enrichment step. In a test using 28 target and non-target strains only the targets were detected and two calibration curves, for pure cultures and artificially contaminated samples, were used to evaluate the efficiencies of this method. Amplification efficiencies of this multiplex real-time PCR were excellent in pure cultures and artificially contaminated shrimps. The limits of detection in artificially contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and 103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial samples and the results were comparable to standard culture methods. This efficient multiplex real-time PCR, where each test takes only 50 min after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products.     

Survey of domestic food purchases and related home handling practices in the Abruzzo region (central Italy): Data collection and analysis through a language-independent classification system

A telephone survey was carried out on 13,486 randomly selected households located in the Abruzzo region (central Italy). Three questionnaires were specifically designed in relation to three different groups of foods (Meat and meat products, Fishery products, Fruit and vegetables). Questions were mainly focused on the amount and number of purchases, type of retailer, and home food handling practices with respect to the week prior to the interview. Data were classified according to a multilingual thesaurus system (LanguaL). Results allowed to estimate domestic purchases (in kg) per capita for different food categories. The category “Red meat/Cattle” accounted for a large part of purchases in the “meat and meat products” group (10.9 kg per capita/year; 32.2% of purchases in the group) while the category “Fish or related organism/Fish” was the most purchased in the “fishery products” group (6.9 kg per capita/year; 63.3%). The aggregation of more detailed characteristics enabled the identification of popular categories of foods, such as “Red meat/Cattle/Divided into pieces” (25% of the “meat and meat products” group). Significant differences (p < 0.05) were found between the four different provinces (L'Aquila, Chieti, Pescara and Teramo) with respect to “Poultry/Chicken” and “Red meat/Cattle” categories. Householders were also asked about post-purchase food handling practices that might be hazardous to food safety. More than 20% of those surveyed stated that they thawed frozen meat at room temperature. The degree of doneness after cooking of different food categories was generally high: over 90% of products purchased in the majority of meat and fish categories were properly cooked. However a noticeable proportion of householders (about 15%) reported medium or rare cooking of “Red meat/Cattle” and “Red meat/Swine”. Differences (p < 0.05) were also found between consumers of different ages, with people over 65 years old being more prone to freeze meat and cook it thoroughly.The survey was carried out in a specific geographic area and on a statistically significant sample of households, thus allowing a collection of data on domestic habits relating to food purchases and home food handling practices. This information should be included in the framework of quantitative microbial risk assessment (QMRA) models as a measure of the actual exposure of consumers to pathogenic microorganism. The LanguaL system also proved to be a practical language-independent method useful not only to identify and describe food items but also to classify them according to specific food safety characteristics.     

Development of a novel target-enriched multiplex PCR (Tem-PCR) assay for simultaneous detection of five foodborne pathogens

In the present study, a novel target-enriched multiplex PCR (Tem-PCR) assay was developed for simultaneous detection of Salmonella spp., Listeria (L.) monocytogenes, Staphylococcus (S.) aureus, Escherichia (E.) coli O157:H7 and Shigella spp.. DNA primers including universal primer and composite primer pairs were used in this Tem-PCR assay. Of which, the universal primer was linked to the 5'-end of each specific primer designed targeting Salmonella invA gene, Staphylococcus aureus femA gene, Shigella ipaH gene, Listeria monocytogenes hly gene and Escherichia coli O157:H7 rfbE gene, respectively, generating the composite primers. During PCR amplification, the composite primer pairs were employed to enrich target genes of different pathogens in initial cycles and the universal primer was employed to enrich the amplicons produced with the composite primers priming in later PCR cycles. Significantly, the Tem-PCR assay overcomes the amplification disparity resulting from primers competition observed in conventional multiplex PCR assay. With the evaluation of the Tem-PCR assay for the detection of these five foodborne pathogens, the assay showed high specificity to the target bacteria with an analytical detection limit of <2.0 × 10² CFU/mL for each, and practical detection capability, suggesting a novel multiplex PCR method for detecting foodborne pathogens.     

石化设备风险评估系统研究进展

风险评估是对生产系统的危险性进行定性和定量分析,评估该系统发生危险的可能性、造成的损失及其严重程度。它以安全管理和科学决策为基础,用专家经验和计算机的存储与逻辑推理能力来预防评估事故。其评估的目的是让设备管理者掌握设备的完好程度和提前了解设备的危险地段,以便合理地分配维护资金,从而变设备的盲目被动维修为预知性主动维修。文章综述了石化设备风险评估在国内的发展概况、评估方法及内容,指出了目前该评估系统需进一步完善的方面,并对其未来发展作一展望。     

A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean,maize and rice in highly processed products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil.     

A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species

A novel common primer multiplex PCR (CP-M-PCR) was applied to detect four kinds of meats (chicken, cattle, pig and horse) as raw materials. A common adapter was designed in the 5′-end of species-specific reverse primers which matched with the species-specific DNA sequences for each species and also used as the common primer (CP). CP-M-PCR primers were designed to uncover different length fragments of 239, 292, 412, and 451 bp from chicken, cattle, pig and horse meats, respectively. The bands of specific DNA fragments amplified by CP-M-PCR method still appeared until the concentration of species-specific primers diluted to 0.015 pmol and primer sensitivity was increased by 100 times compared with conventional multiplex PCR without CP. CP-M-PCR detection limit of the DNA samples was 0.1 ng (36.4 copies) for single kind of meat as well as four kinds of meats. CP-M-PCR method simplified the PCR reaction system and conquered the disparate amplified efficiency from different primers. The CP-M-PCR method could be widely applied in practical detection for simultaneous identification of other meat species and their products.     

A multiplex real-time PCR method for the quantitative determination of equine (horse) fractions in meat products

The recent horse meat scandal that rocked Europe in early 2013 shows how important it is for the routine food control authorities to constantly evolve analytical tools for the accurate evaluation of meat products among others, to ensure that product declaration and actual composition correlate. While in most cases qualitative detection approaches suffice for product evaluation, in other cases a quantitative analysis is important to distinguish between inadvertent contamination and deliberate adulteration.In this work an optimized real-time qPCR-based approach is presented and compared with another real-time based method for the detection of equine sequences among others in meat products. The method is a multiplex system for the simultaneous quantification of horse, beef, pork and sheep fractions, and was validated for use in the routine analysis of meat products. We describe a modular multiplex approach, where a quadruplex system (without sheep) and a pentaplex assay (with an integrated sheep detection system) can be applied in meat detection and quantification strategies. The analysis is matrix independent and relies on concomitant quantification of the animal species present in the food sample against the backdrop of myostatin, a universal sequence present in most mammalian and poultry species. The limit of detection of the analytical method was 10 genome copy equivalents. For validation of the method, meat samples comprising differing meat compositions were analysed, and the assay performed well in terms of robustness and reproducibility.     

Application of FINS and multiplex PCR for detecting genuine abalone products

Abalones rank first on the list of the four treasures from the sea in Chinese cuisine. It is regarded as a tonic for yin-enrichment and nourishing the lungs and liver. Hong Kong is a major entrepôt of abalones, importing 48% of the global production volume. Rapid detection of substitutes in abalone products is essential for safeguarding consumer interests and ensuring proper delivery of healthcare. Two molecular tools are applied to authenticate abalone products. Firstly, forensically informative nucleotide sequencing (FINS) analysis based on mitochondrial 16S rDNA region of abalone products retailed in Hong Kong revealed that all sliced and one canned products matched the sequences of other gastropods but not Haliotis species, suggesting the presence of substitutions. Secondly, multiplex PCR with a Haliotis-specific primer was used to facilitate rapid and reliable exclusion of substitutes from abalone products.     

Risk-based microbiological criteria for Campylobacter in broiler meat: A comparison of two approaches

Risk-based microbiological criteria can offer a tool to control Campylobacter in the broiler meat production chain. Recently two approaches have been applied to derive such criteria and to analyse their potential impact in terms of human health risk reduction: the risk-based version of the established microbiological criteria approach, that applies a microbiological limit (ML) for sample data, and the Danish “case-by- case” risk assessment approach, that applies a limit for the relative risk estimate (relative risk limit, RRL) based on sample data. In this study, data sets from Sweden and Denmark are used to compare the performance of the two approaches in terms of efficiency, i.e. the balance between the residual risk after implementation of the criterion and the percentage of non-complying batches, and the attending uncertainty. The analysis shows that the two approaches are equally efficient, and suggests that the RRL criterion is attended with less uncertainty. The two approaches are compared and their advantages and disadvantages are discussed. Given the uncertainties attending the results of the analysis, more research in terms of data collection, risk assessment and uncertainty analysis would be needed to develop these risk-based criteria further.     

Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 10² CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 10² CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 10³ and 3.6 × 10³ CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.     

A multiplex PCR assay for species identification of raw and cooked bonito

Attempts are made to establish one-step multiplex PCR assay for distinguishing five species of raw and cooked bonito including Euthynnus pelamis, Euthynnus affinis, Auxis rochei, Auxis thazard, and Sarda orientalis. After constructing the 1141 bp complete mitochondrial cytochrome b genes of five bonito species and other five contrastive Scombridae species, five sets of species-specific primer were designed to amplify different cytochrome b gene fragments in each species individually. The amplified lengths of fragments were respectively 143 bp for A. rochei, 236 bp for E. pelamis, 318 bp for A. thazard, 398 bp for E. affinis and 506 bp for S. orientalis, which could be obviously differentiated from each other on DNA electrophoresis. The five sets of species-specific primer were mixed and applied to simultaneously detect bonito species. All species from 12 commercial raw fish and five species out of eight cooked bonito fillets were successfully identified by the multiplex PCR assay. Experiments carried out demonstrate that the multiplex PCR assay was useful for identifying species of non-overheating fish product.     

天然气长输管道的定量风险评价方法

定量风险评价是天然气管道安全管理的有效方法,通过风险评价,可以将管道事故可能造成的灾害风险降低到可接受标准以下。在对天然气长输管线风险分析的基础上,指出造成事故灾害性后果的主要方式是热辐射和冲击波。对天然气管道事故率、事故后果进行了分析,得出了热辐射引起的人员死亡概率的计算方法。天然气管道的风险可以用个人风险和社会风险进行定量评价,介绍了定量计算个人风险和社会风险的方法,并结合实例进行了计算和分析。     

基于失效数据的油气管道定量风险评价方法

油气管道风险评价作为当前的热点问题,逐渐由定性评价向定量评价过渡。为了减少油气管道定量风险评价过程中主观因素的影响,建立了基于管道失效历史数据的油气管道定量风险评价模型,通过分析美国管道与危险品安全管理局(PHMSA)等数据库的管道分类失效数据,确定了油气管道的基本失效概率与修正因子的指标体系,再根据各个指标量化的难易程度将修正因子分为定量、半定量及定性3种类型,其中前两类指标可以量化或量化分级,仅后一类指标依赖于专家的经验判断,大大降低了风险评价的主观依赖性。进一步构建了油气管道风险评价矩阵,用失效概率量化失效可能性,用后果评分量化失效后果的严重程度。最后,将该油气管道定量风险评价方法应用于某输气管线,并绘制"红橙黄蓝"4色管线风险分布图,识别出该管线的高风险管段。结论认为:(1)基于管道失效历史数据的风险评价方法能够客观地量化管道的失效概率,准确进行风险分级,有利于实施风险分级管控策略;(2)建议建立全行业或全国范围的管道失效数据库,以期为基于历史数据的定量风险评价乃至制订风险管控措施提供依据。     

Mathematical quantification of inactivation of Vibrio parahaemolyticus on two types of surface soiled with different substrates

Survivability of foodborne pathogens on food-processing surfaces is an important factor in understanding and quantifying bacterial transfer to foods (i.e. cross-contamination). This study examined the survival of Vibrio parahaemolyticus on two different surfaces in a laboratory-based simulation. V. parahaemolyticus was inoculated onto both polypropylene and stainless steel surfaces following contamination with saline solution (SS), tryptone soya broth (TSB), or seafood purge. V. parahaemolyticus remained viable on polypropylene for 10 days, but was undetectable within 24 h on the stainless steel surface. The survivability was similar on polypropylene in the presence of all contaminating substrates, as shown by data from the Weibull and biphasic models (adjusted-R² > 0.91). However, for stainless steel, SS and TSB prolonged the survival of V. parahaemolyticus to 144 and 120 h, respectively. Survivability data revealed a shoulder period in the first 4 h and a slight tailing effect at the end of the survival curve in the presence of seafood purge, which suggested that the biphasic model might be appropriate (adjusted-R² = 0.9442). These results indicate that the biphasic model may accurately estimate pathogen survival for cross contamination exposure. Integrating the survivability model into quantitative studies will help understand cross contamination.     

套管段井筒完整性风险评价方法研究

井筒完整性评价的目的在于了解井筒风险状况,以便防止井筒结构产生失效破坏。影响井筒完整性的因素包括技术套管完整性、水泥环质量和地层因素3个环节,技术套管完整性是井筒完整性最重要的因素,而水泥环质量不好和地层条件差会增大井筒失效的风险,因此,整个井筒失效是3个环节失效综合的结果。利用层次分析法建立了套管井段井筒完整性的系统模型,给出了井筒完整性评价的整体思路,提出了利用套管监测、预测、测井等手段评价任意井段的井筒完整性方法,给出了对任意井深井筒完整性状态及全口井井筒完整性状态的风险评价模型。     

海洋平台组块吊装装船过程的风险评估方法研究

阐述渤海某海洋石油平台组块吊装上船过程风险评估的整体步骤 ,提出一套具体、实用的系统界定、风险及风险源识别、事故树建立、风险辨识和建立风险评价准则的方案 ;着重说明确定风险辨识和建立风险评价准则的方法和步骤 ,强调这 2点必须由专业技术人员协同完成 ;还给出一种将风险因素定性计算予以量化的方法。     

Development and validation of a micellar liquid chromatography-based method to quantify melamine in swine kidney

Melamine is a toxic compound illegally added to animal feed to falsely boost protein content. This represents a strong threat to consumer's health, as melamine can reach human through the food chain. An easy and reliable micellar liquid chromatography-method was developed to detect melamine in swine kidney. The analyte was extracted by shaking in methanol. Melamine was eluted from the HPLC column without interferences in <8.0 min. Mobile phase was a 0.11 M sodium dodecyl sulfate – 7.5% propanol at pH 3 solution running under isocratic mode through a C18 column at 1 mL/min and absorbance detection at 210 nm. The method was validated according to the Food and Drug Administration guidelines: sensitivity, calibration range (0.3–30 μg/g), linearity (r² > 0.9997), precision (<7.6%), accuracy (−8.3–3.6%), recovery (82.1–92.4%) and robustness (<5.1%). The methodology was applied to swine kidney samples purchased from a local supermarket. The method can be used as a screening method for routine post-mortem diagnose melamine intoxication in animals and detect feeding with poultry containing melamine, in order to withdraw the corresponding flesh. This would be mandatory to prevent consumer intoxication and improve the quality of food.