Robust PCR‐based method for quantification of bovine milk in cheeses made from caprine and ovine milk

To prevent fraud and enhance quality assurance, credible analysis of dairy products is crucial. A common problem is the addition of cheaper bovine milk to caprine and/or ovine dairy products and when not declared addition of bovine milk constitutes fraud. The aim was to develop a rapid, robust and sensitive method for the identification of adulteration of caprine and/or ovine cheeses with bovine milk. New quantitative real‐time polymerase (qPCR) assays were designed for the specific determination of bovine DNA (Cow1) and bovine, caprine and ovine DNA (BoCaOv). These were applied to 17 samples of caprine cheese and 24 of ovine cheese. Results showed that 17% (7/41) of these cheeses contained >5% bovine milk. As bovine milk was not declared as an ingredient in any of the samples, this represents adulteration. Other cheeses that contained detectable bovine milk at ≤5% (22%; 5/41) might pose a health risk to people allergic to bovine milk.     

Rapid DNA preparation from milk and dairy process samples for the detection of bacteria by PCR

In this communication, a rapid, easy pretreatment of milk and dairy process samples is presented in order to detect the microbes present in the samples by the polymerase chain reaction (PCR). It includes the use of commercial membrane cards for the disruption of cells and the storage of DNA, and the inclusion of a card punch in a PCR as the template source. Successful detection of starter, probiotic and pathogen strains in dairy food samples was obtained in PCR by using the membrane-based method for DNA preparation.     

IpO: plasmids and methods for simplified,PCR‐based DNA transplant in yeast

Many yeast experiments require strains modified by recombinant DNA methods. Some experiments require precise insertion of a DNA segment into the genome without a selectable marker remaining. For these applications, we developed a new PCR‐based method for marker‐free DNA transplant. The current PCR‐based method requires the labour‐intensive construction of a PCR template plasmid with repeats of the DNA segment flanking URA3. The design of a new vector, IpO, reduces the work in cloning a single copy of the DNA segment between overlapping URA3 fragments present in the vector. Two PCRs are performed that capture the DNA segment and one or the other URA3 fragment. When the PCR products are co‐transformed into yeast, recombination between the overlapping URA3 fragments restores URA3 and transposes the cloned DNA segment inside out, creating a repeat‐URA3‐repeat cassette. Sequences designed into the PCR primers target integration of the cassette into the genome. Subsequent selection with 5‐fluoro‐orotic acid yields strains that have 'popped out' URA3 via recombination between the DNA repeats, with the result being the precise insertion of the DNA segment minus the selectable marker. An additional advantage of the IpO method is that it eliminates PCR artifacts that can plague the current method's repeat‐containing templates. Copyright © 2014 John Wiley & Sons, Ltd.     

Lyophilisation improves the extraction of PCR‐quality community DNA from pig faecal samples

BACKGROUND: Faeces are increasingly used as sources of DNA for genetic and ecological studies. Although multiple methods to preserve faecal samples prior to DNA extraction have been used (e.g., 70% or absolute ethanol, freezing at ?20 °C or in liquid nitrogen) no information is at present available in the literature on the use of lyophilised faeces. Accordingly, the yield and quality of the community DNA obtained by using four different commercial DNA extraction kits (QIAamp DNA Stool Mini Kit, REALPURE Spin Kit, SPEEDTOOLS Tissue DNA Extraction Kit, and JETQUICK Tissue DNA Spin Kit) from fresh and lyophilised samples of faeces were studied here. RESULTS: The use of lyophilised faeces resulted in a 1.5‐ to 2‐fold increase in DNA recovery relative to the use of fresh faeces regardless of the kit used. Among the four kits tested, the best results were obtained with the QIAamp DNA Stool Mini Kit. Community DNA obtained from lyophilised faeces also provided the best restriction fragment length polymorphism (PCR–RFLP) profiles, which should guarantee a better representation of the microbial diversity present in faecal samples. CONCLUSION: As compared with using fresh faecal samples for pig faecal microbiota studies, lyophilisation improved both DNA yield and quality of the information arising from the PCR–RFLP method of analysis. Copyright © 2009 Society of Chemical Industry     

原料乳中沙门氏菌的快速过滤富集及PCR检测

尽管国内外以PCR技术为基础对食源性致病菌的检测手段已经基本成熟,但是由于大多数有8～12h的预增菌步骤,从而增加了检测时间和检测成本。建立了对原料乳中沙门氏菌的快速、简单、灵敏PCR检测技术。人工污染沙门氏菌的原乳,先离心脱脂,然后添加Na2EDTA获得澄清乳液,最后通过0.45μm微膜过滤收集菌体。整个PCR检测过程可在6.5h以内完成,且检测灵敏度可达到1～10mL-1。该方法值得推广,应用于原乳中该食源致病菌的日常检测。     

Traditional and innovative production methods of Fiore Sardo cheese: a comparison of microflora with a PCR‐culture technique

Fiore Sardo is a cheese manufactured on farms in Sardinia from raw ewe’s milk, with Protected Designation of Origin (PDO). The aim of the study was to evaluate the effects of introducing a whey starter culture and specific mechanised cutting operations (innovative) vs traditional manufacture on the microbiological composition of the cheese, as assessed by conventional and molecular (PCR‐Culture Tecnique) methods. Five batches were manufactured by innovative and traditional methods and there was no significant difference between the cheeses.     

Differentiation of Clostridium perfringens and Clostridium botulinum from non‐toxigenic clostridia,isolated from prepared and frozen foods by PCR‐DAN based methods

During the elaboration process of prepared and frozen foods, Clostridium sp. have been reported. From those microorganisms, C. perfringensand C. botulinum may pose a high risk for the consumers. To avoid these pathogenic organisms an HACCP program should be implemented, but in addition sensitive and moderately time consuming microbiological methods for monitoring C. perfringens and C. boulinum should be established. In this work, an RFLP analysis of the 16S rDNA will be developed to differentiate C. perfringens from other Clostridium sp. In addition, a PCR protocol, will be assayed to detect C. botulinum. Both two methods will be compared with biochemical characterization by API system. The restriction analysis of the 16S rDNA with Taq I and RsaI showed at least the same sensitivity to differentiate C. perfringensfrom clostridial isolates as biochemical identification. However, the former method takes only 8–10 h of analysis as compared with 24–48 h required for biochemical characterization. With the specific PCR protocol to detect C. botulinum a band of 1.1 kbp was obtained derived from the specific amplification of BoNT genes, taking 6–8 h for analysis. Both two molecular DNA based methods should be considered as verification techniques of pathogenic clostridia in the HACCP program.     

Automated DNA preparation from maize tissues and food samples suitable for real-time PCR detection of native genes

There is an increasing need for high-throughput analyses of plants and food samples for the presence of specific DNA sequences, e.g. transgenic contaminations. We developed and optimized conditions for the automated isolation of DNA from several maize tissues and various edibles containing maize using the MagNA Pure LC system (Roche Applied Science). Our results show that the system provided is capable of isolating DNA from any tested source. Quantification of an endogenous gene by LightCycler real-time PCR revealed that the DNA is suitable in quality and quantity for multiple PCR analyses.     

An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica

A molecular method for the detection of Salmonella enterica strains based on 16S rRNA sequence analysis was developed by a modification of the previously described PCR primer 16SFI [J. Appl. Bacteriol. 80 (1996) 659], which was combined with a newly developed primer annealing at the position 66-82. Only approximately two thirds of now determined Salmonella 16S rRNA sequences contained a region identical to the 16SFI primer sequence and the reverse primer 16SIII was also not specific. Combined, these two primers have been claimed to allow the specific detection of all Salmonella; however, in this study, they did not recognize S. bongori and 3 out of 78 tested S. enterica strains. They also identified some of the tested Enterobacter cloacae strains as Salmonella. On the contrary, the new primer pair, MINf and MINr, made it possible to recognize correctly all of the 78 tested S. enterica strains, representing 31 different Salmonella serovars. None of the 23 non-Salmonella strains from the related gamma-proteobacterial genera was incorrectly recognized as belonging to S. enterica.     

An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces

There is a need for standardised, robust, reproducible molecular and culture methods to achieve clarification of the inactivation of Mycobacterium avium subsp. paratuberculosis (Map), the causative microbial agent of Johne's disease, in (faecally) contaminated milk and other food products such as meat. This study assessed the performance of a commercially available Map DNA extraction kit for milk Adiapure and accompanying PCR detection kit Adiavet alongside 'in-house' molecular and culture methods in an inter-laboratory ring trial using raw milk spiked with Map-infected faeces. The combined Adiapure-Adiavet Map DNA extraction and detection kit consistently detected 30 copies of IS900 (equivalent to approximately 2 cells) ml(-1) raw milk, when used in four different laboratories. Improvements in sensitivity and ease of use for 'in-house' Map detection were observed when the Adiapure extraction kit was combined with 'in-house' detection assays. Detection by real-time PCR methods, using the commercial extraction and detection systems, resulted in an overall detection rate of 100%, 90%, 85% and 25% for respective Map concentrations of 300, 30, 3 and 0.3 copies of IS900ml(-1) raw milk. Map, at 300 copies of IS900 (equivalent to approximately 20 Map cells) ml(-1) raw milk, was recovered from all samples cultured in mycobacteria growth indicator tube (MGIT) medium, from 10 of 12 samples on Herrold's egg yolk medium (HEYM) and not recovered from any samples using BACTEC medium. In conclusion, the Adiapure DNA extraction kit allows for sensitive and easy detection of Map in raw milk. The extraction method can form a candidate part of essential methodology and real-time PCR can further increase the sensitivity of the detection method. Moreover, MGIT medium is promising for culture-dependent detection of Map from raw milk.     

An improved PCR primer pair based on 16S rDNA for the specific detection of Salmonella serovars in food samples

Salmonella serovars are some of the major bacterial pathogens that can cause sporadic cases and outbreaks of foodborne illness. Based on the sequence data in the V3 region of the 16S rRNA gene, two PCR primer pairs have been designed for the detection of all serovars of Salmonella. However, none of these primers were specific for Salmonella because complete sequence homology with certain non-Salmonella strains has been found within each of them. Thus, the specificities of these two primer pairs could not rely on only one of the two primers. In this study, we modified our previous 16SFI primer by extending one base at the 5' end and three bases at the 3' end. The modified primer, 16S-Sal, was designed with one or more mismatched bases near the 3' end of the primer annealing to the corresponding sequences of non-Salmonella strains. Such modification eliminates interference from Citrobacter freundii and Enterobacter cloacae as occurs with the 16SFI primer. When 16S-Sal and a degenerate primer, 16S-CCR, were used as a primer pair, detection specificity of Salmonella serovars was achieved. Because this primer pair was used for PCR detection of the salmonellae in food samples, such as whole milk and chicken meat, as low as 1 to 9 CFU/g (ml) of the food sample could be detected when a 8-h preculture step was performed prior to the PCR. For chicken meat, the endogenous microflora did not interfere with the PCR results.     

Application of magnetic polymethacrylate‐based microspheres for the isolation of DNA from raw vegetables and processed foods of plant origin

A method for the microextraction of DNA from raw vegetables and highly processed foods of plant origin suitable for PCR analysis was developed. It is based on non‐selective binding of DNA in the presence of PEG 6,000/NaCl to hydrophilic magnetic nonporous poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) ‐ P(HEMA‐co‐GMA) microspheres covered by carboxyl groups. The quantitative polymerase chain reaction (qPCR) by in‐house designed primers targeting a highly repetitive rDNA locus allowed for detection of plant DNAs in a wide range of concentrations (0.1 pg/μL to 10 ng/μL). The described procedure is fast and simple. We further demonstrate that relatively mild acidic treatment of vegetable at elevated temperatures resulted in a dramatic reduction of PCR efficiency indicating extensive degradation of DNA during pickling. The described method is suitable for the analysis of highly degraded DNA from pickled food products.

Practical applications

DNA‐based methods, such as the polymerase chain reaction (PCR), represent an important genetic approach for identification of plant species composition of foods. DNA from processed foods varies in both quality and quantity between the protocols used. Plant samples are generally characterised by a complex composition containing various inhibitors of PCR. Current methods have been tailored for specific samples while no universal protocol is available. Development of a simple universal procedure leading to PCR‐ready DNA would be beneficial. Isolation strategies based on solid phase systems have become popular for PCR‐ready DNA isolations from complex matrices. Here we applied magnetic hydrophilic P(HEMA‐co‐GMA) microspheres to extract DNA from raw vegetables and highly processed foods of plant origin. Proposed small scale PCR‐ready DNA extraction protocol was comparable with a silica‐based DNeasy Plant Mini Kit (Qiagen) and classical CTAB extraction methods in quality and amplificability of DNA while it is less costly and time consuming.     

Formulation of an oat‐based fermented product and its comparison with yoghurt

In an attempt to develop a fermented, non‐dairy product based on oats, a new kind of oat base, Adavena® M40, was fermented with two different yoghurt cultures. Adavena® M40 is a concentrated liquid (with a dry matter content of 16 or 18%) derived entirely from oats, with maltose as the main carbohydrate source and an intact β‐glucan content. The oat base was heat treated for 5 min at 85 °C prior to inoculation. Additives in the form of stabiliser, fat and flavours were used. Texture, syneresis, colour and sensory parameters were evaluated. Yoghurt was used as a control. The final product had an acidity and viscosity similar to those of yoghurt. Addition of xanthan gum (0.03% w/v) improved the texture and overall appearance of the product. The product had the same texture as yoghurt but showed less syneresis. The mixture was less white than the control. The oat‐based, yoghurt‐like product showed high acceptability in terms of acidity, texture and overall appearance. The addition of flavours resulted in a higher acceptance of the final products by the panellists. The β‐glucan content was still high after the fermentation process. The results indicated the potential for a new, fermentable, oat‐based product with high acceptance and a high final β‐glucan content. © 2001 Society of Chemical Industry     

An improved plate culture procedure for the rapid detection of beer‐spoilage lactic acid bacteria

Lactic acid bacteria (LAB) are the most frequently encountered beer‐spoilage bacteria, and they can render beer undrinkable owing to the production of lactic acid, diacetyl and turbidity. Three beer‐spoilage strains, 2011–6, 2011–8 and 2011–11, were isolated from finished beers. Based on the 16S rRNA sequence analysis, these three isolates were identified as Lactobacillus acetotolerans. Only the horA homologue was detected in these strains, while the horC homologue was not detected. In addition, an improved plate culture method for the rapid detection of beer‐spoilage LAB by the addition of catalase was evaluated. Supplementation with catalase enhanced the growth and colony sizes of the spoilage LAB investigated. These beer‐spoilage bacteria, including some slowly growing strains, were detected within five days of incubation using the modified method. Taken together, the modified procedure could be a rapid countermeasure against beer‐spoilage LAB, and it compared favourably with the conventional plate culture method. Copyright © 2014 The Institute of Brewing & Distilling     

Enzymatic hydrolysis combined with high‐pressure homogenisation for the preparation of polysaccharide‐based nanoparticles from the by‐product of Flammulina velutipes

In this study, protease, α‐amylase and 5‰ β‐glucanase enzymolysis in combination with high‐pressure homogenisation were used for the preparation of polysaccharide‐based nanoparticles from Flammulina velutipes stipes, respectively, named FNP‐1, FNP‐2 and FNP‐3, and the nanoparticles were subsequently characterised. The FNP size distribution ranged from 50 nm to 300 nm, among which FNP‐2 and FNP‐3 were smaller than FNP‐1, based on the SEM images. GC‐MS results showed that these particles were mainly composed of glucose and glucosamine. The FNP dispersions at 1 wt% behaved as non‐Newtonian, shear‐thinning fluids, and the FNP‐3 dispersion presented superior viscoelasticity. With an increasing degree of enzymolysis, the thermal stability of the FNPs decreased. In addition, these particles presented various cation‐exchange properties. Therefore, the Flammulina velutipes polysaccharide‐based nanoparticles obtained from this study can be potentially used as a promising functional food ingredient in the food industry.     

Development and application of a new multiplex real‐time PCR assay for simultaneous identification of Brucella melitensis,Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese

Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes are important foodborne pathogens in milk and milk products, which are responsible for a variety of diseases that pose serious hazards to public health and food safety. The objective of this study was to develop a novel multiplex RTi‐PCR for the detection of B. melitensis, C. sakazakii and L. monocytogenes and to characterise the potential risk of these pathogens in raw milk and cheese. The raw milk (n = 25) and cheese samples (n = 20) were analysed by multiplex RTi‐PCR assay and detected for quantification of the three pathogens. In this study, B. melitensis, C. sakazakii and L. monocytogenes were simultaneously identified using BMEII0466, mms operon and hly as target genes, respectively. The multiplex RTi‐PCR assay that was developed showed good sensitivity and selectivity for the pathogenic microorganisms (r² = 0.986–0.997). Multiplex RTi‐PCR results showed that most of the samples were contaminated with the pathogens screened.