Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.     

Application of polymerase chain reaction–restriction fragment length polymorphism analysis and lab‐on‐a‐chip capillary electrophoresis for the specific identification of game and domestic meats

BACKGROUND: The objective of this study was to adapt and improve previously published polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods aimed at the identification of game and domestic meats, by replacing the gel electrophoretic steps for DNA fragment analysis by a chip‐based capillary electrophoresis system. RESULTS: PCR amplification of a mitochondrial 12S rRNA gene fragment and subsequent digestion of the amplicons with either MseI or a combination of MboII, BslI, and ApoI endonucleases generated characteristic PCR‐RFLP profiles that allowed discrimination among ten relevant game and domestic meat species. The Agilent 2100 Bioanalyzer utilises capillary electrophoresis on a microchip device that is capable of rapidly sizing DNA fragments, offering a valuable recent development for the analysis of complex DNA banding patterns. CONCLUSION: Results obtained in this work indicated that banding resolution on the system was sensitive, with detection of some small DNA fragments that were not observed with the published conventional PCR‐RFLP gel‐based method. Therefore, the new faster and easy handling procedure provides an additional powerful tool that can be employed for meat speciation. Copyright © 2009 Society of Chemical Industry     

Genetic differentiation between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR–RFLP analysis of a 12S rRNA gene fragment

PCR–RFLP analysis was applied to the identification of two closely related flatfish species: sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Amplification of DNA isolated from fish muscle samples was carried out using a set of primers flanking a region of 321 base pairs (bp) from the mitochondrial 12S rRNA gene. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for AciI and MwoI endonucleases. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with AciI and MwoI enzymes allowed the unequivocal identification of sole and Greenland halibut species. © 2000 Society of Chemical Industry     

Molecular identification of five commercial flatfish species by PCR–RFLP analysis of a 12S rRNA gene fragment

Refrigerated or frozen fillets of commercial flatfish species are sometimes mislabelled, and identification of those products is needed to avoid fraudulent substitution. Molecular identification of five commercial flatfish species (order Pleuronectiformes), ie Lepidorhombus whiffiagonis (megrim), Platichthys flesus (flounder), Reinhardtius hippoglossoides (Greenland halibut), Scophthalmus maximus (turbot) and Solea vulgaris (= S solea) (sole), has been carried out on the basis of the amplification of an approximately 433 bp segment from the mitochondrial 12S rRNA gene using the polymerase chain reaction (PCR) and universal primers. Direct DNA sequencing from two PCR products for each flatfish species was carried out, and sequences were used to select six restriction enzymes. PCR products of 15 individuals of each species were cut with each enzyme, resulting in species‐specific restriction fragment length polymorphism (RFLP). The five flatfish species could be identified by application of the restriction enzyme AluI as well as by using different combinations of a pair of enzymes, ie DdeI and either AciI or MwoI. No intraspecific genetic polymorphism was found for any of the six enzymes. Results confirmed the usefulness of this technique to distinguish and genetically characterise refrigerated or frozen pieces of these five flatfish species. Copyright © 2003 Society of Chemical Industry     

PCR‐Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR‐amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five‐base (ScrFI) and two four‐base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR‐RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP‐PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.     

Distribution of Candida in the oral cavity and its differentiation based on the internally transcribed spacer (ITS) regions of rDNA

This study aimed to determine the distribution of Candida species in the oral cavity and differentiate the species based on PCR amplification, including HinfI and MspI digestion, in order to assess the effectiveness of using the rDNA region for species identification. Samples from saliva as well as palate, tongue and cheek mucosa surfaces were collected from 45 individuals, consisting of three groups: periodontal disease patients; denture‐wearers; and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for colony‐forming units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction, based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI‐5.8S‐ITSII PCR product was digested by HinfI and MspI restriction enzymes. The microbial loads on all sites of the denture‐wearers were found to be significantly higher than control, while in the periodontal disease group only the microbial loads on the tongue were significantly higher than control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to those of seven control species, allowing the differentiation of all seven species and the identification of 14 species from the clinical samples. The MspI restriction digest was not able to distinguish between C. albicans and C. dubliniensis, whereas the HinfI digest could not distinguish between C. tropicalis and C. parapsilosis. It was concluded that PCR–RFLP of the candidal rDNA region has potential for species identification. This study demonstrates the potential use of candidal rDNA as a means for identifying Candida species, based on genotype. The results also indicate the possibility of constructing genetic probes that target specific restriction fragments in the ITSI‐5.8S‐ITSII region, enabling swift and precise identification of Candida species. Copyright © 2012 John Wiley & Sons, Ltd.     

Species identification of red and brown seaweeds using ITS ribosomal DNA amplification and RFLP patterns

The identity of five macroalgae used as sea vegetables or food ingredients has been determined by amplification of the ribosomal DNA ITS region and RFLP analysis. This allows the detection of specificity patterns for each species and provides an alternative method, when morphological or biochemical methods fail, for control of their use as food ingredients. Alga‐specific PCR primers have been used to determine the ITS rDNA sequences of DNAs extracted from dried and ground algae up to 5 years old. The seaweeds studied were the red algae Palmaria palmata (dulse), Porphyra umbilicalis (nori), and Chondrus crispus (pioca) and the brown algae Himanthalia elongata (sea spaghetti) and Laminaria sp (konbu). Total genomic DNA suitable for amplification was extracted from the alga powder following the CTAB method. This methodology allowed the sequencing of the amplified product and the drawing of theoretical restriction maps useful in the choice of restriction enzymes for RFLP analysis. Enzymes that appeared useful included Mbo I and Alu I. Cutting with a single enzyme was sufficient to obtain characteristic patterns for the red algae P palmata, P umbilicalis and C crispus. For the two brown algae H elongata and Laminaria sp the ITS rDNA sequence showed a lack of suitable restriction site, contrary to other species for which characteristic patterns were obtained. © 2003 Society of Chemical Industry     

Identification of gadoid species (Pisces, Gadidae) by sequencing and PCR–RFLP analysis of mitochondrial 12S and 16S rRNA gene fragments

We applied PCR–RFLP (Polymerase Chain Reaction – Restriction Fragment Length Polymorphism) analysis to identify seven gadoid species of different biogeographical origin and commercial relevance, namely Gadus morhua (Atlantic ocean); Trisopterus minutus capelanus, Trisopterus minutus minutus, Molva elongata, Phycis blennoides, Micromesistius poutassou (Atlantic ocean and Mediterranean sea); Theragra chalcogramma (Pacific ocean). Two DNA fragments belonging to mitochondrial 12S and 16S rRNA genes of about 430 and 630 bp, respectively, were isolated by PCR amplification. Their direct sequencing showed a significant genotypic diversity among gadoid species, useful for species identification. Digestion of 16S rRNA gene PCR fragment with MvaI or Bsh1285I restriction enzymes, followed by agarose gel electrophoresis of the cleaved products, yielded specific restriction profiles that enabled direct, visual identification of the species analyzed. This PCR–RFLP method allowed a clear and rapid discrimination of the gadoid species studied.     

Detection of pig derivatives in food products for halal authentication by polymerase chain reaction –restriction fragment length polymorphism

A method for detection of the presence of pig derivatives in three types of food products—sausages and casings, bread and biscuits—using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene was developed. Genomic DNA of sausages and casings, bread and biscuits were extracted. The genomic DNA from the food products were found to be of good quality for the sausages and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs (bp). However, no genomic DNA was detected from the casing samples and poor quality of genomic DNA was extracted from bread and biscuits. No amplification of mt cyt b gene was produced from bread and biscuit samples. To differentiate between samples, the amplified PCR products were digested with restriction enzyme (RE) BsaJI, resulting in species‐specific RFLP. The cyt b PCR‐RFLP species identification assay gave excellent results for detection of pork adulteration in food products and is a potentially reliable technique to avoid species adulteration or fraudulent species substitution for halal authentication. Copyright © 2006 Society of Chemical Industry     

Combination of SYBR Green II and TaqMan Probe in the adulteration detection of Dendrobium devonianum by fluorescent quantitative PCR

‘Zipi Fengdou’ is a restorative food made of the stem of Dendrobium devonianum, Paxt., whose high commercial value presents the constant risk of adulteration with cheaper ‘Foudou’ products made from other Dendrobium species. Therefore, this study developed two assays capable of detecting D. devonianum based on SYBR Green II and TaqMan probe real‐time PCR. By performing the diagnostic real‐time PCR based on SYBR Green II, two DNA fragments (163 and 159 bp) were specifically amplified from the internal transcribed spacer (ITS) region of D. devonianum. The average cycle threshold (C_t) values of two D. devonianum samples collected from different Chinese provinces were shown to be 16.65 for ITS1‐4 and 16.38 for ITS2‐1, which were significantly (P < 0.001, SPSS) different from those of the reference Dendrobium species used as adulterants of ‘Zipi Fengdou’ (34.94 for ITS1‐4, 34.67 for ITS2‐1). Moreover, in the assay based on TaqMan probe real‐time PCR, two TaqMan probes were designed and tested for the quantitative detection of D. devonianum in commercial samples labelled as ‘Zipi Fengdou’. As a result, this assay specifically and sensitively distinguished the processed Fengdou’ sample of D. devonianum from those of adulterant Dendrobium species.     

PCR‐RFLP and DAMD‐PCR genotyping for Salvia species

BACKGROUND: Identification of genotypes in Salvia is complicated owing to the morphological similarity and common occurrence of natural hybridisation within Salvia species. Species‐ and genotype‐specific DNA markers are very useful for plant identification, breeding and preservation programmes and can also provide a general overview on the prediction of plant essential oil yield. RESULTS: Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) was used for identification of species‐specific chloroplast and mitochondrial organelle DNA markers, and directed amplification of minisatellite DNA polymerase chain reaction (DAMD‐PCR) was used for genotyping of plant materials. Application of PCR‐RFLP resulted in species‐specific DNA markers, and use of DAMD‐PCR resulted in reproducible DNA patterns that are useful in Salvia genetic studies. Multivariate cluster analysis and principal coordinate analysis indicated that there were relationships between DNA marker patterns and essential oil yields at the species level. CONCLUSION: Results showed that genetic variations in Salvia are wide, and DNA patterns of relatedness among plant species appeared to correlate with essential oil yields. Further studies are required to confirm the application of PCR‐RFLP and DAMD‐PCR markers for selection of Salvia species with higher essential oil yield. Copyright © 2008 Society of Chemical Industry     

Comparison and evaluation of molecular methods used for identification and discrimination of lactic acid bacteria

BACKGROUND: Lactobacillus and Bifidobacterium strains are present in a great variety of habitats, including fermented products, probiotic concoctions and the human colon. Some species are so closely related that it is difficult to distinguish them by microbiological techniques. Nevertheless, discrimination of isolates is an important issue in respect of application, and molecular methods such as restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) or species‐specific polymerase chain reaction (PCR) might help in resolving this problem. In this study, PCR, RFLP and sequencing were applied to identify lactobacilli and bifidobacteria originating from various sources and the DSMZ strain collection. RESULTS: The microbiological composition of foods was analysed by molecular methods. Using species‐specific PCR primers, three restriction enzymes (AluI, HhaI and RsaI) and sequencing, three Bifidobacterium and six Lactobacillus reference strains could be distinguished and four additional lactobacilli of food origin were identified. CONCLUSION: A combination of three molecular methods resulted in successful discrimination of nine reference strains and four isolates of food origin. Since these methods are not always accurate owing to their high genetic homogeneity, it is advisable to use more than one method for the identification of L. casei and closely related species. Copyright © 2012 Society of Chemical Industry     

Development of a Polymerase Chain Reaction System for the Detection of Dog and Cat Meat in Meat Mixtures and Animal Feed

The identification of species origin of meat represents a considerable problem for food and animal feed analysis. In the present study a PCR‐mediated method for the detection of dog and cat meat was developed. For this the cytochrome b gene sequence of both species was analyzed by restriction fragment length polymorphism (RFLP) analysis. The use of the restriction enzymes Alu I and Hae III yielded specific restriction profiles characteristic for each species. The meat of both species could additionally be differentiated with species‐specific oligonucleotide primers based on specific parts of the cytochrome b gene sequences characteristic for dog and cat. The use of these oligonucleotide primers allowed a direct identification of dog and cat meat in meat mixtures even after heat treatment.     

基于ITS2序列及二级结构对紫苏与其变种、紫苏子与其混伪品的鉴别

采用ITS2 一级序列联合其二级结构对紫苏与其变种、紫苏子与其混伪品进行鉴定,为紫苏药材的使用提供更科学的分类方法.收集紫苏与其近缘种、紫苏子混伪品基源植物样本,提取DNA进行PCR扩增;并结合GenBank中的序列获得所有物种的ITS2序列.应用MEGA7.0软件进行序列分析、计算种内种间遗传距离、构建NJ树,利用I...     

Molecular identification of yeast species associated with 'Hamei'--a traditional starter used for rice wine production in Manipur, India

In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called 'Hamei' is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four 'Hamei' samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database. ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with 'Hamei' were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1-K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from different samples. Other karyotypes K2-K7 were very unique with less than 80% similarity. Finally using mitochondrial DNA restriction analysis (mt-DNA RFLP), S. cerevisiae strains belonging to the major karyotype K1 were distinctly differentiated with highly polymorphic bands by Hinf I and Hae III endonucleases.     

Genetic Identification of Lophius budegassa and L. piscatorius by PCR-RFLP Analysis of a Mitochondrial tRNAGlu/Cytochrome bSegment

Identification of Lophius budegassa(black‐bellied angler) and L. piscatorius(angler) (Lophiiformes) was carried out on the amplification of a 486 bp tRNA^Glu/cytochrome b segment using the polymerase chain reaction (PCR). Direct DNA sequencing of 6 PCR products was carried out. Six restriction endonucleases (AluI, CfoI, HaeIII, HinfI, Mae, and ScrFI) with different species‐specific restriction fragment length polymorphism (RFLP) were selected. Digestions of PCR products from 30 individuals showed no intraspecific polymorphism. Double digestions (CfoI and HinfI, and HaeIII and ScrFI) were simpler and more rapid than single digestions. This technique is suitable for distinguishing tails of both Lophius species.     

Metabolism of the Hepatotoxic Compound Sophoraflavanone G in Rat Liver Microsomes

Our study aimed at investigating the metabolic characteristics of sophoraflavanone G (SFG), one of the hepatotoxic constituents of Sophora flavescens, in rat liver microsomes (RLMs). SFG was metabolized to 3 phase I metabolites, di‐hydroxylated SFG (M1), mono‐hydroxylated SFG (M2), dehydrogenated product of mono‐hydroxylated SFG (M3) and 3 SFG glucuronides (M4, M5, and M6) by RLMs. The formation kinetics of M2 conformed to biphasic kinetics in RLMs. The formation kinetics of M4 and M5 best‐fitted the Hill equation kinetics. Chemical inhibition studies found that CYP1A2 and CYP2E1 were the major enzymes responsible for the formation of M2, and the formation of M4 and M5 may be catalyzed by multiple UGT1A isoforms.     

Molecular approaches for the detection of truffle species in processed food products

Molecular identification techniques were applied in order to analyse food products containing fragments of some Tuber species. Samples of fungal DNA were processed by analyses of the internal transcribed spacer (ITS) region. The polymerase chain reaction (PCR) using truffle species‐specific primers, multiplex PCR, restriction fragment length polymorphism (RFLP) analysis, sequencing of the ITS region and specific oligonucleotide probe hybridisation were used. The results obtained demonstrate the applicability of these molecular strategies to the identification of truffles, even when their morphological characteristics are difficult to interpret owing to the drastic treatments utilised in food preparation or the use of unripe fruit bodies (lacking spores). Furthermore, testing was also possible starting from very small amounts of sample and degraded DNA. The methods described have important applications in both the production and sale of such food products, in order to avoid fraud and reveal the possible presence of other fungal species. © 2002 Society of Chemical Industry     

Biotechnological characterisation of exocellular proteases produced by enological Hanseniaspora isolates

Twenty‐six enological Hanseniaspora isolates were identified by ITS PCR‐RFLP and D1/D2 domain of 26S rDNA sequencing and assayed for exocellular protease production. Based on qualitative data, six isolates, belonging to H. guilliermondii, H. valbyensis and H. occidentalis species, were selected to continue the study. Analytical procedure was optimised, and protease activities were quantified and characterised on the basis of different biotechnological factors. Protease activity was quite glucose, fructose and ethanol content independent, but divalent cation affects activity; these data support that they were aspartic proteases. The effect of 2‐mercaptoethanol suggests the importance of disulphide bonds to maintain the structure of the active centre. Our results show these enzymes could be suitable for using in biotechnological processes at neutral pH, such as cheese, bread and meat industries. This is, to our knowledge, the first work showing the induction process to induce and characterise proteolytic activity in Hanseniaspora isolates.     

Rapid PCR-RFLP Method for the Identification of Marine Fish Fillets (Seabass, Seabream, Umbrine, and Dentex)

A rapid and reliable PCR‐RFLP method was optimized to identify marine fish fillets. Seabass, seabream, umbrine, and dentex were considered in the study. After DNA extraction and PCR, the 359 bp amplification products, obtained from gene encoding the cytochrome b, were subjected to restriction enzyme analysis. All the enzymes tested were not able to distinguish all the 5 species at the same time, but the combination of the results obtained from the digestion HaeIII and NlaIII can be used to differentiate the fish fillets considered. The method described is sensitive, rapid, and reliable, and it could be used to expose fraudulent substitutions with less valuable fish.