Occurrence of mycotoxigenic fungi and mycotoxins in animal feed from bihar,india

A total of 387 samples comprising animal feed, poultry feed and cattle cakes from India were examined in order to isolate mycotoxin-producing fungi as well as mycotoxins. Of 385 Aspergillus flavus group isolates 53% were capable of producing aflatoxins in liquid SMKY medium. Toxigenic strains of other mycotoxigenic fungi, viz A ochraceus, A versicolor, Penicillium citrinum and Fusarium moniliforme, were also recorded. In feed samples, beside the aflatoxins present in 139 samples zearalenone, ochratoxin A and citrinin were also recorded alone or as cocontaminants. A monsoon climate together with the socioeconomic conditions of this region are important determinants for the high incidence of mycotoxins in animal food.     

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.     

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.     

多重实时荧光定量PCR检测肴肉中的特定腐败菌

本试验以真空包装水晶肴肉中的3株特定腐败菌屎肠球菌(Enterococcus faecium)、中间耶尔森菌(Yersinia intermedia)和清酒乳杆菌(Lactobacillus sakei)为研究对象,通过克隆3株菌的特异性DNA片段,构建重组质粒作为阳性模板,建立一种多重实时荧光定量PCR方法同时检测这3株特定腐败菌,结果表明:建立的多重实时荧光定量PCR具有较高的特异性和灵敏度,同时还具有很好的稳定性,可用于真空包装水晶肴肉贮藏期间特定腐败菌数量的检测,从而可以间接评价产品的货架期。     

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R² > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.     

Quantitative detection of Secale cereale by real-time PCR amplification

Wheat, barley, rye free diet is required to control coeliac disease and therefore adequate analytical tools are necessary to check gluten-free products. At the moment, official analytical methods are based on immunochemical assays for gliadin detection. However, DNA-based methods for detection of specific sequences have been extensively proposed as alternative to the protein-based ones, e.g. for transgenic contamination analysis. The aim of this work has been to develop an alternative quantification method for rye-content evaluation in raw materials and processed food based on real-time PCR approach using both SYBR green detection system and TaqMan fluorogenic probe.     

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log₁₀/300 μl C. jejuni cells, one log₁₀ better (lower) than that of IMS-qPCR (2.1 log₁₀ CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log₁₀ CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.     

Taqman real-time PCR for the detection and enumeration of Saccharomyces cerevisiae in wine

Saccharomyces cerevisiae is the main yeast species responsible for wine fermentation; however, its presence during maturing or barrel-ageing can sometimes result in a reduction in the quality of wine by refermentation. In this work, we developed a quantitative real-time PCR (QPCR) for the rapid detection and quantification of S. cerevisiae in wine. The primers and the hydrolysis probe (TaqMan^®) were designed from the sequence of a DNA fragment present only in S. cerevisiae and absent in other wine yeasts obtained from an RAPD-PCR analysis. The QPCR developed was highly reproducible, allowing the specific detection and quantification of this yeast in artificially contaminated wines, with a detection limit of 78 CFU/mL. Furthermore, the usefulness of the QPCR developed was evaluated through the quantification of the yeast in wine samples obtained from vineyards, confirming the quantitative capacity of the method. The methodology developed was specific, fast and a sensitive tool for the detection and enumeration of S. cerevisiae cells in wine.     

SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs

A real-time polymerase chain reaction assay using primers targeting the porcine-specific mitochondrial 12S rRNA gene and universal eukaryotic primers amplifying a conserved fragment of the nuclear 18S rRNA gene has been developed for the detection and quantification of porcine DNA in food and feedstuffs. The 18S rRNA primers were used as endogenous control for the total content of PCR-amplifiable DNA in the sample. The assay was tested on DNA extracted from raw and heat-treated binary mixtures of porcine tissues in a plant matrix, and on DNA extracted from reference feedstuff samples. Analysis of experimental mixtures demonstrated the suitability of the assay for the detection and quantification of porcine DNA in mixtures containing as little as 0.1%.     

A novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting Enterobacteriaceae in the dairy and other food industries

The presence of lactose-fermenting Enterobacteriaceae and coliforms is routinely assessed to determine the hygienic quality of water and foods, particularly dairy products. This paper reports the use of lacZ-specific primers in an SYBR green I-based real-time PCR method for the easy and rapid detection of coliforms in dairy products. A large number of bacterial species were assayed to establish the specificity of the method. The sensitivity of the method was assessed using artificially contaminated cheeses. The limit of detection was 1 coliform cell in cheese samples enriched for 8 h in a culture medium. The entire procedure, including sample processing, enrichment, DNA extraction, and real-time PCR amplification, can be completed within 10 to 12 h, making it a single-day assay.     

Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

Vibrio parahaemolyticus is found in aquatic environments and is the leading cause of gastroenteritis due to seafood consumption worldwide. We evaluated a quantitative real-time PCR (Q-PCR) assay with hydrolysis probes, to determine whether this method could be used for the efficient counting of total, tdh and trh-positive V. parahaemolyticus in shrimps. We assessed the specificity of this assay, using 62 strains from 12 non target bacterial species of the Vibrio, Photobacterium, Shewanella and Aeromonas genera. Only V. parahaemolyticus with the appropriate target gene generated a fluorescent signal. We assessed the robustness of this assay, by analyzing spiked shrimps by Q-PCR and traditional culture methods, using most probable number (MPN)-PCR. After a 6 h enrichment period, the assay successfully detected total and pathogenic V. parahaemolyticus in shrimps samples spiked with less than 5 V. parahaemolyticus cells/g of shrimp. The Q-PCR results obtained were compared with those obtained by most probable number (MPN)-PCR format. An excellent correlation between the two methods was observed in all cases (R² > 0.9742). The application of this Q-PCR assay to 85 natural shrimp samples also resulted in the successful quantification of V. parahaemolyticus in this matrix, and the counts obtained were correlated with those obtained by (MPN)-PCR (P = 0.2598). Thus, this rapid and sensitive Q-PCR can be used to quantify V. parahaemolyticus in natural shrimp samples. This assay could be proposed, in response to the demands of the European Commission, as a tool for testing for the presence of vibrios in crustaceans, making it possible to legislate in this domain.     

Multiplex real-time PCR for the detection and quantification of DNA from four transgenic soy Mon89788, A5547-127, Roundup Ready, A2704-12 and lectin

In the past, the transgenic soybean Roundup Ready was the predominantly cultivated transgenic soybean. With the announcement of Monsanto Corp. that this trait will be replaced by Mon89788 and the release of other company??s transgenic soy crops, this unique status of one trait has ended. Therefore, a multiplex quantitative real-time PCR system was developed and characterized for three additional transgenic traits Mon89788, A2704-12 and A5547-127. It showed amplification efficiency, correlation and sensitivity similar to the single PCR systems applied therein. This system allows relative multiplex quantification and/or delta?Cdelta Ct method and therefore an efficient control of products during production, trade and sale.     

实时荧光定量技术在食品微生物检测和研究中的应用

实时荧光定量PCR技术是通过检测PCR产物中荧光讯号强度来达到定量的目的,不仅实现了对核酸信息量的分析比较,而且与常规PCR相比,它具有特异性更强、能有效解决PCR污染问题、自动化程度高等特点。文章概述了实时荧光定量PCR技术的原理、优缺点及其在食品微生物检测中的应用与研究进展,并探讨了它的技术发展和应用前景。     

Multiplex real-time PCR for the simultaneous detection and quantification of DNA from three transgenic rice species and construction and application of an artificial oligonucleotide as reference molecule

According to the EU and Swiss legislation for food, only approved traits of transgene plants are allowed to be imported and sold to the consumer. In order to control imports of rice and rice products from retailers, dealers and importing companies, efficient and reliable methods for the detection and quantification are a prerequisite. Therefore, a novel pentaplex real-time polymerase chain reaction system was developed and validated for the quantitative determination of three genetically modified rice lines at once. This system simultaneously determines DNA contents of the phospholipase-gen, a rice species specific gene, the 35S:BAR-construct, as the promotor of different transgene rice lines and the specific systems for LL62, LL601 and Bt-63-rice (Shanyou63). The test exhibits a good specificity and sensitivity for the transgenes in the range of 0.01–1%. It proved its efficiency and reliability in daily routine. Due to the lack of appropriate reference material for the Bt-63-rice, a reference oligonucleotide was artificially constructed. This oligonucleotide proved its applicability in diagnostic analysis.     

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.     

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.     

实时荧光PCR定性定量检测混合食用油脂中的花生油成分

为鉴别鉴定花生油在食用油脂中的存在与否及其含量,研究了定性定量检测花生特异性基因片段的分子生物学检测方法。结果表明,生花生需加热处理后再提取DNA才能作为PCR扩增的模板,食用油脂中可提取出用于定性定量PCR的DNA,花生的Arah1基因是具有花生种属特异性的基因,采用实时荧光PCR方法检测Arah1基因片段可定性定量检测混合食用油脂中的花生油成分。     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.