Development of a novel multi-locus variable-number tandem repeat analysis (MLVA) combined with high resolution melting analysis (HRMA) for Listeria innocua typing and its application in a food processing plant

Listeria monocytogenes is the causative bacteria of listeriosis, a type of food poisoning with high mortality, which mainly infects people through the consumption of ready-to-eat food. Most cases of L. monocytogenes contamination have occurred at food processing plants. Therefore, food companies must carefully monitor the proliferation and spread of this bacterium. Listeria innocua has often been reported to exist in food processing plants when the presence of the genus Listeria is investigated. In addition, because L. innocua and L. monocytogenes are known to act together, L. innocua is an important hygiene indicator for food companies. Multilocus variable-number tandem repeat analysis (MLVA) is a method used to discriminate between strains, based on the discrepancies in the number of repeating units within the tandem repeats (TRs). In many cases, MLVA uses capillary electrophoresis (CE) or base sequence analysis. However, the equipment used for these analyses is expensive, and requires a certain level of knowledge and skill for operation. Therefore, it is only used at research facilities. However, in order to rapidly trace the sources of contamination in food processing plants, it is necessary to easily discriminate between strains within the working areas. Therefore, in this study, we established a simpler MLVA method, using high resolution melting analysis (HRMA) to analyze the diversity of variable-number of tandem repeats (VNTRs). We investigated three VNTR loci (used in a previous MLVA-CE analysis) using MLVA-HRMA. The MLVA-HRMA of 23 L. innocua strains showed that this method differentiates between strains at a more or less equal level with MLVA, which uses base sequence analysis. HRMA is also used to investigate gene polymorphisms based on the Tm value of the PCR products. Therefore, a different peak pattern is observed depending on the internal SNP even if the number of repeating units are the same. We then evaluated the efficacy of MLVA-HRMA in differentiating between 96 strains isolated at food processing plants. This analysis demonstrated the usefulness of the MLVA-HRMA method and its discriminatory ability in tracing the sources of contamination in food processing plants. Since this method is simpler, and cheaper than the existing MLVA methods, it could see widespread use in food processing plants as a rapid investigative tool.     

The application of Bar-HRM (Barcode DNA-High Resolution Melting) analysis for authenticity testing and quantitative detection of bean crops (Leguminosae) without prior DNA purification

The Leguminoseae family includes several economically important genera like legumes. Legumes are the second most important agricultural family after cereals in terms of harvested area and production. Legumes are key elements in the Mediterranean diet and as winter annuals with nitrogen fixing capacity, they are extremely important for sustainable agriculture worldwide.We report here the application of the Bar-HRM (Barcode DNA High Resolution Melting) analysis method with the universal plant DNA barcoding region trnL which allowed the identification, adulteration and quantification of major Greek and Mediterranean in general bean species. Bar-HRM detected Lupinus spp. adulterants in Glycine max flour as low as 1:100. Moreover, the method was coupled with the rapid Phire PCR kit that does not require prior DNA purification. This makes the method a very fast and effective tool for barcoding Legumes and particularly for the crops examined not only for their authenticity but for quantitative detection of purity of their seeds or their processed food and feed products.     

High resolution melting analysis of a COI mini-barcode as a new approach for Penaeidae shrimp species discrimination

Penaeidae family includes shrimp species of high commercial value, sharing noticeable morphological similarities, which makes them potential targets of adulteration. Therefore, mechanisms for authentication and certification of such crustaceans, frequently included in processed foods, constitute a benefit for the food industry. Litopenaeus vannamei, Penaeus monodon, Fenneropenaeus indicus, Metapenaeus affinis and Melicertus kerathurus are some of the most relevant penaeid shrimps, being their differentiation of high importance. This work intended to develop a new approach for the specific detection and differentiation of those five closely related shrimp species based on high resolution melting (HRM) analysis targeting a cytochrome oxidase subunit I (COI) mini-barcode. The method enabled the differentiation of the five species with high levels of confidence (>99%), being successfully applied to analyse processed seafood samples. F. indicus and L. vannamei were the main identified species in the commercial products. When verifying labelling compliance, four samples suggest adulterations based on the complete or partial substitution of declared species. The proposed method proved to be a potential tool for the rapid and cost-effective differentiation of penaied shrimp species.     

Towards the development of a multi-element analysis by ICP-oa-TOF-MS for tracing the geographical origin of processed tomato products

In this preliminary work, the mineral composition (Li, Be, Al, V, Cr, Mn, Co, Cu, Zn, Ga, As, Rb, Sr, Ag, Cd, In, Cs, Ba, Tl, Pb, Bi, and U) of forty-one processed tomato samples, coming from three different countries (Italy, China and Spain), was investigated by Inductively Coupled Plasma orthogonal acceleration Time-of-Flight Mass Spectrometer (ICP-oa-TOF-MS), exploiting the characteristic of simultaneity of the measurements that is distinctive of this type of instrument. The large amount of data achieved through multi-element analysis was statistically processed by Linear Discriminant Analysis (LDA), and a good geographical origin discrimination of tomato samples (99.9% of cases correctly classified) was obtained. Li and Sr could distinguish Chinese from EU samples, while Co and Cu were able to differentiate Spanish tomato from the other ones. Moreover some elemental ratios (Li/Cr, Mn/Cu, Co/Cu and Sr/Cd) allowed a better clusters separation than single elements.The work is still in progress, but preliminary results indicates that ICP-oa-TOF-MS could be considered a fast, precise and accurate instrument for multi-element determinations to be used for tracing tomato provenance.     

Discriminant analysis for unveiling the origin of roasted coffee samples: A tool for quality control of coffee related products

Coffee quality is highly dependent on geographical factors. Based on the chemical characterization of 25 coffee samples from worldwide provenances and same roasting degree, Discriminant Analysis (DA) was employed to develop models that are able to identify the continental or country (Brazil) provenance of blind coffee samples. These models are based on coffee composition, particularly on several key compounds either with or without significant impact on aroma, such as 2,3-butanedione, 2,3-pentanedione, 2-methylbutanal and 2-ethyl-6-methylpyrazine. All models were validated with new and independent data from literature, and also through cross validation and permutation tests. Furthermore, the robustness of the proposed models in case of incomplete characterization data was also tested, being concluded that missing data is supportable by the models. In the whole, this article provides compelling arguments for the development of DA-based tools with the purpose of controlling the quality of coffee in terms of their continental and/or national origins.     

Effects of sediment,seawater, and season on multi-element fingerprints of Manila clam (Ruditapes philippinarum) for authenticity identification

The objective of this study was to select suitable elements which reflect the link with geographical origin while are less affected by season to build robust discriminant models for the protection of Jiaozhou Bay clam by the multi-element fingerprinting technique. Manila clam (Ruditapes philippinarum), sediment, and seawater samples were collected from three major clam producing areas of China (Jiaozhou Bay, Nantong coast, and Dalian coast) in May and September 2014. The concentrations of 25 elements (Na, Mg, Al, etc.) were determined in all the samples. The data obtained was analyzed by analysis of variance, Pearson correlation, a radar plot, and stepwise linear discriminant analysis. The results demonstrated that the models built on five elements (Mg, Cd, Sn, Ba, Ce) are correlated with geographical origin and independent of seasonal variations, and can be used to completely differentiate Jiaozhou Bay clams from the samples from Nantong and Dalian.     

Verifying the geographical origin of poultry: The application of stable isotope and trace element (SITE) analysis

Stable isotope and elemental analysis, together with statistical processing of the resultant data has been used to determine the geographical origin of poultry and hence provide a means to verify poultry labels originating from major producing countries/regions. Multivariate statistical analysis has demonstrated that 18 variables, including carbon, hydrogen and nitrogen stable isotope ratios and elemental concentrations of magnesium, thallium, rubidium and molybdenum, are important parameters in poultry origin determination. Using cross-validated discriminant analysis 88.3% of poultry geographical origins were correctly classified (n = 339). Individual correct-classification rates were as follows; China, 100% (n = 36); Brazil, 94.1% (n = 101); Europe 92% (n = 87); Chile 82.6% (n = 46); Thailand, 70.3% (n = 46) and Argentina 50% (n = 10). The main identification errors were associated with miss-classification of Argentinean samples with those originating from Chile and Thailand. Carbon stable isotope ratios of chicken meat indicate the quantity of maize in the diet and this leads to useful discrimination between a large proportion of European poultry and poultry reared in locations such as South America, Thailand and China where maize feeding predominates. The use of poultry carbon isotope values as a simple ‘screening’ parameter to differentiate European poultry meat from other major importers is not as reliable as for the differentiation of European and South American beef. However carbon isotope ratios will be useful in most instances to corroborate suspicion of mislabelling of non corn-fed European poultry. The stable isotopes of hydrogen and oxygen in chicken meat change in a similar way to surface waters around the globe. Our findings support the hypothesis that the global isotopic variation of stable isotopes in drinking water and feed are transferred into animal tissue and can be used to help establish an animal's geographic origin. This is a significant finding and mirrors our observations for beef skeletal muscle δ²H ‰ and δ¹⁸O ‰ values. These systematic variations can be exploited to give a ‘low-resolution’ indication of an animal's geographic origin (e.g. Northern Europe versus the tropics).     

The classification of sea cucumber (Apostichopus japonicus) according to region of origin using multi-element analysis and pattern recognition techniques

Element composition fingerprints were investigated for their potential to classify sea cucumber (Apostichopus japonicus) samples according to their geographical origin. Fifteen elements in 39 sea cucumber samples from three water environments (Bohai Sea, Yellow Sea, East China Sea) of China were quantitatively analyzed by inductively coupled plasma-mass spectrometry (ICP-MS). Pattern recognition techniques including principal component analysis (PCA), cluster analysis (CA, squared Euclidean distance between objects and between-groups linkage method as a clustering procedure) and linear discriminate analysis (LDA) were used for this purpose. Fifteen elements (Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Hg and Pb) in the sea cucumber samples of different regions showed significant differences (P < 0.05). Furthermore, sea cucumber samples from three water environments were accurately differentiated by PCA and CA. LDA gave an overall correct classification rate of 100.0% and cross-validation rate of 100.0%. These results demonstrate the usefulness of the multi-element analysis combined with pattern recognition techniques for authenticating of the geographical origin of sea cucumber in China.     

Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification

Pork identification in four types of food products, which are sausages and the casings, bread and biscuits, using species-specific polymerase chain reaction (PCR) detection of a conserved region in the mitochondrial (mt) 12S ribosomal RNA (rRNA) gene was developed. Genomic DNA of the food products were successfully extracted except for the casing samples, where no genomic DNA was detected. The extracted genomic DNA was then subjected to PCR amplification targeting the specific regions of the 12S rRNA gene. The genomic DNA from the food products were found to be of good quality and produced clear PCR products on the amplification of 12S rRNA gene of 387 base pairs (bp) from pork species. The species-specific PCR identification yielded excellent results for identification of pork derivatives in food products and it is a potentially reliable and suitable technique in routine food analysis for halal certification.     

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.     

节能增效的碳分子筛(CMS)生产技术

天科股份宁夏分公司建立CMS生产过程质量控制技术和采用仪器监控高温炭化、沉积、活化过程,有效调整孔结构,实现节能增效"一步法"新工艺。摆脱当前国内CMS生产过程无控制和活化、沉积过程耗能、费时、耗体力的"多步法"落后生产工艺。为提升CMS产品质量水平,降低生产成本,获得良好经济效益,提供了实实在在的生产技术。     

压裂用高效发泡助排剂JX-YL的性能评价与应用

针对萨南油田南二、三区西块地层压力低,且原油含蜡量高的特点,在压裂施工中应用了JX—YL高效发泡助排剂。对压裂用高效发泡助排剂JX—YL的性能进行了室内评价,介绍了JX—YL的作用原理及现场应用情况。JX—YL释放的热量可以解除高粘油区块由于低温液体的注入所造成的蜡质、胶质的伤害;生成的大量惰性气体,可以有效提高地层返排压力;产生的大量泡沫,可有效降低压裂液的滤失;表面活性减小了毛细管的阻力。室内与现场试验结果表明,JX—YL具有返排快、溶蜡降粘、助排能力强的性能,比普通压裂液初期返排率提高25％左右,有效减小了压裂液对地层的伤害,取得了明显的增产效果及显的经济效益。     

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL⁻¹ and 100 ng mL⁻¹ of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g⁻¹ (110–120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.     

稳定高分辨率感应测井的浅探测拟合曲线和对阵列感应测井质量的评价

阵列感应测井的目标之一就是定量计算冲洗带电阻率(Rxo).多阵列感应测井仪可产生一条非常准确的深聚焦电阻率测井曲线,该曲线接近于地层真电阻率(Rt).然而,浅聚焦电阻率测井曲线,尤其是10-in.探测深度的电阻率测井曲线,在地层电导率与邻层电导率之间对比变化大时不十分可靠.阵列感应测井的基本聚焦算法建立在博恩近似法的基础上.博恩近似法是通过博恩几何因子建立感应测量值与地层电导率分布的线性关系.因此,两个因素可造成浅聚焦电阻率测井曲线中的不稳定,即浅聚焦和深阵列感应之间的非线性关系和通过软件聚焦处理的深阵列感应测井到浅聚焦测井的误差传播.几种先进的方法,如反演和基于非均质背景的聚焦(IBF),能阐述这个问题.这两种方法能够在复杂环境中提取地层和冲洗带电导率.然而,这两种算法都要求高强度的计算并且需要正演模拟计算.由于新一代贝克*阿特拉斯聚焦型测井系统的发展,一个具有6个子阵列的感应测井仪轻松获得投产.文中给出一种简单、高效的方法来改善浅聚焦电阻率测井,例如高分辨率感应测井(HDIL)的10-in.探测深度测井曲线.当用一个新的6阵列聚焦滤波产生的10-in.探测深度电阻率测井曲线时,该方法要去除HDIL中的最长的子阵列.对现行的HDIL处理系统做最少量的修改,这种方法就可完成实时处理.聚焦型阵列感应测井仪和HDIL测井仪的质量控制处理过程都是针对井下校正测量进行的,总结如下第一,根据测量的原理识别资料的相容模式;第二,用一种称为"模式分解"的技术将测量值与相容模式对比;第三,根据相容模式去除不正确数据,重新构建测量值;最后,通过对比原始测量值和重构建测量值来评价数据质量.     

煤层气井完井用PE筛管的地质适应性分析

煤层气井水平井段易垮塌是导致煤层气开发过程中产量难以提升的重要原因之一,出于煤层气开采之后的采煤作业安全考虑,金属材质的筛管被禁止下入煤层,采用PE筛管完井则成为解决上述问题的重要途径。为分析PE筛管能否适用于易垮塌的煤层中,利用断裂力学方法开展了煤层的井壁失稳机理分析,并基于时间延迟效应原理分析了近井壁地带的煤岩裂纹尖端应力强度因子,提出了在孔隙压力压降漏斗存在的情况下裂纹延展范围的判定方法。通过实例计算分析了煤岩垮塌可能对PE筛管造成的最大压力。利用室内单轴压缩机模拟大块煤岩掉落时PE筛管（外径50.8 mm,壁厚4.6 mm）单轴受压变形工况,得到了管体抗挤性能和PE管体挤压破坏判别标准;以实验结果为基础,采用数值模拟软件分析了直径分别为50.8、63.5、76.2、88.9、101.6 mm筛管的抗挤性能,从而优选出了不同尺寸筛管的合理壁厚,对煤层气井完井用的PE筛管的研制和应用具有一定的指导意义。