Temporal Temperature Gel Electrophoresis Research Articles

Analysis of the bacterial diversity in Moroccan Jben cheese using TTGE, DGGE, and 16S rRNA sequencing.

This research investigated the physicochemical, microbiological, and bacterial diversity of Jben cheese, a popular artisanal variety in Morocco. The bacterial diversity was explored using culture-independent methods, including temporal temperature gel electrophoresis (TTGE), denaturing gradient gel electrophoresis (DGGE), and high-throughput sequencing (HTS). Significant intra-sample differences were observed for most physicochemical parameters within each milk type, while inter-sample differences occurred between cow and goat cheeses for dry matter and ash. Jben cheese exhibited distinct characteristics, with low pH values of 3.96, 4.16, and 4.18 for cow, goat, and mixed cheeses, respectively. Goat cheeses had higher fat (49.23 g/100 g), ash (1.91 g/100 g), and dry matter (36.39 g/100 g) than cow cheeses. All cheeses displayed high microbial counts, with a notable prevalence of the lactic acid bacteria (LAB) group, averaging 8.80 ± 0.92 log CFU/g. Jben cheese also displayed high contamination levels with total coliforms, faecal coliforms, yeast, and molds. Fatty acid profiling revealed fraudulent practices in Jben cheese marketing, with cow or mixed cheeses sold as goat cheese, as proven by low capric acid concentration. HTS analysis of Jben cheese identified ten genera and twenty-four species, highlighting Lactococcus lactis as predominant. TTGE and DGGE confirmed the presence of L. lactis but failed to provide the detailed profile achieved through HTS analysis. HTS has been demonstrated to be more reliable, whereas TTGE/DGGE methods, though informative, were more time-consuming and less reliable. Despite limitations, the combined use of TTGE, DGGE, and HTS provided a comprehensive view of indigenous bacterial communities in Jben cheese, identifying L. lactis as the main species.

Supplementation of Diets With Spirulina Influences Immune and Gut Function in Dogs.

Spirulina refers to two species of blue green algae (Arthrospira platensis, and A. maxima) consumed by humans as food for centuries. While, Spirulina has been shown to have immune enhancing properties in several animal and human studies, there are no systematic studies in dogs. The aim of this study was to evaluate the immunomodulatory effect of dietary supplementation with Spirulina in dogs. The study was conducted in two phases: Pre-test (8 wks.) and Test (42 wks.). Thirty adult dogs (mean 2.9 yrs.) were randomized into two groups and fed a nutritionally complete diet in the “Pre-test” phase. At the end of “Pre-test” phase all dogs received a rabies vaccine, and dogs in “test group” were switched to diet supplemented with dried Arthrospira platensis (Spirulina). Response to rabies vaccine was evaluated by Rapid Fluorescent Focus Inhibition Test (RFFIT). Gut immune response was assessed by measuring fecal IgA. Gut microbiota was evaluated by Temporal Temperature Gel Electrophoresis (TTGE) methodology. Repeated measures ANOVA was used to test for differences between groups and statistical significance considered to be p < 0.05. Dogs fed diets supplemented with Spirulina demonstrated enhanced immune status by showing significantly higher vaccine response and higher levels of fecal IgA as compared to the control group. Supplementing diets with Spirulina also resulted in significantly increased gut microbiota stability in the test group. In conclusion, diets supplemented with Spirulina significantly enhanced immune response and gut health in dogs.

Microbiological characterization using combined culture dependent and independent approaches of Casizolu pasta filata cheese.

Casizolu is a traditional Sardinian (Italy) pasta filata cheese made with cow raw milk belonging to Sardo-Modicana and/or Bruno-Sarda breeds added with natural whey starter. This work aims to describe the traditional technology of this product and to evaluate the microbial groups/species involved in the first month of ripening. Raw milk, curd after stretching and Casizolu cheese samples from two different farmsteads were subjected to enumeration of microbial groups, isolation and genotypic characterization of isolates and PCR temporal temperature gel electrophoresis (TTGE) analysis. The counts of lactobacilli and lactococci groups in raw milk were about 5-6 log UFC ml(-1) of milk. These counts tended to increase in curd and cheeses, reaching values higher than 8 log UFC g(-1) of cheese. Culture dependent and independent approaches employed in this work highlighted the fundamental role of Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Lactobacillus paracasei in the manufacture and ripening of Casizolu cheese. Other species frequently isolated were Enterococcus durans, Enterococcus faecium, Enterococcus italicus while Enterococcus lactis, Streptococcus parauberis, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus fermentum and Lactococcus raffinolactis were isolated occasionally. Lactococcus lactis subsp. lactis, Strep. thermophilus and Lact. paracasei were the principal bacterial species involved in the Casizolu cheese manufacturing and ripening. For the first time, Ent. italicus and Ent. lactis were isolated in the pasta filata cheese. This study shows the first data on microbial groups and species involved in the manufacture of Casizolu cheese and highlights the role of Lact. paracasei and Enterococcus spp. from the earliest stages of ripening cheese; furthermore, provides evidence that raw milk cheese is a source of new strains and therefore a reservoir of microbial biodiversity.

Dynamics of the surgical microbiota along the cardiothoracic surgery pathway.

Human skin associated microbiota are increasingly described by culture-independent methods that showed an unexpected diversity with variation correlated with several pathologies. A role of microbiota disequilibrium in infection occurrence is hypothesized, particularly in surgical site infections. We study the diversities of operative site microbiota and its dynamics during surgical pathway of patients undergoing coronary-artery by-pass graft (CABG). Pre-, per-, and post-operative samples were collected from 25 patients: skin before the surgery, superficially and deeply during the intervention, and healing tissues. Bacterial diversity was assessed by DNA fingerprint using 16S rRNA gene PCR and Temporal Temperature Gel Electrophoresis (TTGE). The diversity of Operational Taxonomic Units (OTUs) at the surgical site was analyzed according to the stage of surgery. From all patients and samples, we identified 147 different OTUs belonging to the 6 phyla Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Cyanobacteria, and Fusobacteria. High variations were observed among patients but common themes can be observed. The Firmicutes dominated quantitatively but were largely encompassed by the Proteobacteria regarding the OTUs diversity. The genera Propionibacterium and Staphylococcus predominated on the preoperative skin, whereas very diverse Proteobacteria appeared selected in peri-operative samples. The resilience in scar skin was partial with depletion in Actinobacteria and Firmicutes and increase of Gram-negative bacteria. Finally, the thoracic operative site presents an unexpected bacterial diversity, which is partially common to skin microbiota but presents particular dynamics. We described a complex bacterial community that gathers pathobionts and bacteria deemed to be environmental, opportunistic pathogens and non-pathogenic bacteria. These data stress to consider surgical microbiota as a “pathobiome” rather than a reservoir of individual potential pathogens.

Propionibacterium acnes populations involved in deep pathological samples and their dynamics along the cardiac surgical pathway.

Propionibacterium acnes belongs to the normal skin microbiota, but it is also responsible for acne vulgaris and causes serious infections such as endocarditis and surgical site infections (SSI). The P. acnes population is structured into phylogenetic groups, with phylotype I being associated with acne. Herein, we explore the link between phylotypes and clinical origins in a collection of P. acnes isolated from different body sites, involved in deep infections or healthcare-associated infections (HAI), with particular emphasis on strains from cardiac SSI. Cardiac SSI have been further studied in terms of P. acnes population dynamics during the care pathway. The recA and tly genes phylotypes were compared to hemolytic behavior, susceptibility to antimicrobial agents, and clinical origins. An original approach of recA polymerase chain reaction temporal temperature gel electrophoresis (PCR-TTGE) was developed and applied for the direct identification of P. acnes phylotypes in surgical samples, in order to assess their temporal dynamics during the surgical course. Our results underlined the preferential involvement of IA-2/IB and II phylogroups in HAI and SSI. Unlike IA and II, type IA-2/IB presented a gradual increase with the depth of sampling in the peroperative phase of cardiac surgery. Phylotypes IA and IA-2/IB were both predominant in scar tissues and on postoperative skin, suggesting a specific predisposition to recolonize skin. Particular association of the phylotype IA-2/IB with SSI and its propensity to colonize wounds in cardiac surgery was observed. We assumed that the follow-up of P. acnes phylotypes during pathological processes could give new clues for P. acnes pathogenicity.

Gut microbiota in preterm infants with gross blood in stools: A prospective, controlled study

ObjectiveGross blood in stools is a peculiar entity in preterm infants, but little is known about its etiology. As gut microbiota can be distorted in preterm infants, we aimed to evaluate the gut microbiota in infants with gross blood in stools. Study designIn a prospective, controlled, single-center study, we enrolled all infants born before 34weeks of gestational age presenting gross blood in stools that was either completely isolated or associated with mild clinical symptoms or radiological signs. Each case was paired with two controls who were hospitalized in the same unit and were matched for gestational age and birth weight. The diversity of the gut microbiota was analyzed using 16S rRNA gene PCR and temporal temperature gel electrophoresis. We calculated a diversity score corresponding to the number of operational taxonomic units present in the microbiota. ResultsThirty-three preterm infants with gross blood in stools were matched with 57 controls. Clinical characteristics were similar in cases and controls. There was no statistically significant difference in the diversity score between the two groups, but microbiota composition differed. The proportion of infants with Escherichia coli was significantly higher in cases than in controls (p=0.045) and the opposite pattern occurred for Staphylococcus sp. (p=0.047). ConclusionDysbiosis could be a risk factor for gross blood in stools in preterm infants. Additional, larger studies are needed to confirm the implications of the presence of different genotypes of E. coli and to evaluate preventive actions such as the prophylactic use of probiotics and/or prebiotics.

Supplementation of diets with bovine colostrum influences immune function in dogs

While the need for colostrum in neonates is well established, the systemic effect of feeding bovine colostrum (BC) to adult humans is gaining increasing attention. However, no systematic studies evaluating the immunomodulatory effect of BC in dogs have been reported. The aim of the present study was to evaluate the immunomodulatory effect of dietary supplementation of BC in dogs. The study was conducted in two phases: pre-test (8 weeks) and test (40 weeks), with twenty-four dogs (mean age 2.5 years) randomised into two groups. In the 'pre-test' phase, both groups were fed a nutritionally complete diet. At the end of the 'pre-test' phase, all dogs received a canine distemper virus (CDV) vaccine, and dogs in the 'test group' were switched to a diet supplemented with 0.1% spray-dried BC. Response to the CDV vaccine was evaluated by measuring vaccine-specific plasma IgG levels. Gut-associated lymphoid tissue response was assessed by measuring faecal IgA levels. Gut microbiota were evaluated by the temporal temperature gel electrophoresis methodology. Dogs fed the BC-supplemented diet demonstrated a significantly higher vaccine response and higher levels of faecal IgA when compared with the control group. Supplementing diets with BC also resulted in significantly increased gut microbiota diversity and stability in the test group. In conclusion, diets supplemented with BC significantly influence immune response in dogs.

Distribution and diversity of arbuscular mycorrhizal fungi in grapevines from production vineyards along the eastern Adriatic coast

The colonisation and diversity of arbuscular mycorrhizal fungi (AMF) on roots of grapevines were investigated in production vineyards located along a 500-km-long stretch of karst along the coast of the Adriatic Sea. AMF communities on roots of grapevines were analysed using temporal temperature gel electrophoresis and sequencing of the 18S and internal transcribed spacer segments of the rDNA operon. The AMF colonisation of these grapevines roots was consistent along the whole of this east Adriatic karst region, at 64 to 82% of fine roots. The comparison of the AMF communities on the roots of these grapevines showed that the fungal community associated with grapevine roots seems to be relatively stable, with inter-vineyard variability comparable to intra-vineyard variability. Some of the changes in the fungal communities were attributed to environmental factors (plant-available P) and location of the vineyard, although the latter could also have been influenced by an unmeasured environmental factor. A total of 27 taxa of fungi were identified, including taxa from Glomus group B, based on the sequencing of 18S rDNA. Sequencing of the internal transcribed spacer rDNA yielded 30 different fungal taxa, which comprised eight different Glomeromycota taxa, including Glomus sinuosum and Glomus indicum. To our knowledge, this is the first report of grapevine colonisation by G. indicum.

Reverse transcription quantitative PCR revealed persistency of thermophilic lactic acid bacteria metabolic activity until the end of the ripening of Emmental cheese

For Emmental manufacture two kinds of adjunct culture are added: (i) thermophilic lactic acid bacteria (starters) such as Lactobacillus helveticus (LH), and Streptococcus thermophilus (ST) growing the first day of the manufacture and (ii) ripening culture. ST and LH have a key role in curd acidification and proteolysis at the beginning of the manufacture but are considered to be lyzed for a great part of them at the ripening step. The aim of this work was to assess the metabolic activity of these bacteria throughout manufacture and ripening. During Emmental cheesemaking, LH and ST were subjected to i) population quantification by numerations and by quantitative PCR (qPCR) ii) reverse transcription (RT) Temporal Temperature Gel Electrophoresis (TTGE) iii) transcript quantification by RT-qPCR targeting 16S rRNA, tuf and groL mRNAs to evaluate bacterial metabolic activity. During ripening, ST and LH numerations showed a 2.5 log 10 loss of culturability whereas qPCR on pelleted cells revealed only one log 10 of decrease for both of these species. 10 9 ST and 10 8 LH cells/g of cheese still remained. They contained a stable number of 16S transcript and at least 10 6 copies of mRNAs per 10 9 cells until the end of ripening. These results prove the unexpected persistency of thermophilic lactic acid bacteria starters (ST and LH) metabolic activity until the end of ripening and open new perspectives in term of their involvement in the quality of cheeses during ripening.

A fermented formula in pre-term infants: clinical tolerance, gut microbiota, down-regulation of faecal calprotectin and up-regulation of faecal secretory IgA

Intestinal bacterial colonisation in pre-term infants is delayed compared with full-term infants, leading to an increased risk of gastrointestinal disease. Modulation of colonisation through dietary supplementation with probiotics or prebiotics could decrease such a risk. The present study evaluated clinical tolerance, the effects on gut microbiota, and inflammatory and immunological mucosal responses to an infant formula adapted for pre-term infants that included in its manufacturing process a fermentation step with two probiotic strains, Bifidobacterium breve C50 and Streptococcus thermophilus 065, inactivated by heat at the end of the process. A total of fifty-eight infants (gestational age: 30-35 weeks), fed either the fermented pre-term formula or a standard pre-term formula, were followed up during their hospital stay. Clinical tolerance, faecal microbiota using a culture and a culture-independent method (temporal temperature gel electrophoresis), faecal calprotectin and secretory IgA were analysed weekly. No difference was observed regarding anthropometric data and digestive tolerance, except for abdominal distension, the incidence of which was lower in infants fed the fermented formula for 2 weeks. Bacterial colonisation was not modified by the type of feeding, particularly for bifidobacteria. Faecal calprotectin was significantly lower in infants fed the fermented formula for 2 weeks, and secretory IgA increased with both mother's milk and the fermented formula. The fermented formula was well tolerated and did not significantly modulate the bacterial colonisation but had benefits on inflammatory and immune markers, which might be related to some features of gastrointestinal tolerance.

Medulloblastoma Harbor Somatic Mitochondrial DNA Mutations in the D-loop Region

Despite the growing knowledge on molecular risk factors of the most common malignant brain tumor in childhood, medulloblastoma, its biology remains only partially understood. A previous study investigating the entire mitochondrial genome of medulloblastoma revealed a number of somatic mutations in tumor and corresponding cerebrospinal fluid samples. In our present study we sought to corroborate these results on somatic and germ line mutations by comparing the complete mitochondrial genome sequences of medulloblastoma tissue in a further cohort of patients. Analysis of the entire mitochondrial genome by temporal temperature gel electrophoresis and direct sequencing revealed 6 somatic mutations in 6 of 15 medulloblastoma. All changes were insertions, deletions, or substitutions restricted to the np 303 to 315 poly-C tract of the D-loop region. Three were changes from heteroplasmy to homoplasmy. Two were changes from heteroplasmy to heteroplasmy and one mutation represented a change from homoplasmy to heteroplasmy. In addition, 25 distinct germ line variations were identified. These results are in support of our previous findings on frequency of somatic mitochondrial mutations in medulloblastoma. Somatic alterations were found only in the hypervariable D-loop region, supporting the idea that these control regions contain hot spots for both, germ line variations and somatic alterations of the mitochondrial genome.

Optimized PCR-Temporal Temperature Gel Electrophoresis compared to cultivation to assess diversity of gut microbiota in neonates

Temporal Temperature Gel Electrophoresis of amplified 16S rRNA gene sequences (16S rDNA PCR-TTGE) constitutes a culture-independent molecular method used to study bacterial communities. All the technical steps are crucial for quality and exhaustiveness of the results obtained by such approach. Careful optimization of the protocols used is ideally needed for each ecosystem studied. We present here the strategy used to construct an optimized protocol for a 16S rDNA PCR-TTGE-based analysis of gut microflora in neonates. Improvement of the different steps, i.e. total DNA extraction, amplification in terms of efficiency and reduction of heteroduplex formation, TTGE migration conditions and bacterial identification from TTGE patterns, was performed. The optimized protocol was used for the subsequent analysis of 14 stool samples comparatively to a culture-based method. We showed that a specifically designed ladder representative of the diversity of the studied microflora is a useful tool for the identification of bacterial taxa despite biases inherent to 16S rRNA genes, including intra-genomic heterogeneity. Cultivation and PCR-TTGE gave congruent results but cultivation was more efficient for the detection of minor populations whereas PCR-TTGE gave a more complete description of the major populations. Finally, we demonstrated the reliability, the detection sensitivity and the convenience of the optimized 16S rDNA PCR-TTGE method compared with cultural approaches for studying the premature neonate gut microbiota.

Analysis of β Globin Mutations in the Indian Population: Presence of Rare and Novel Mutations and Region Wise Heterogeneity.

β-thalassaemia is a major public health problem in India. A comprehensive data base of the spectrum of mutations causing β thalassaemia in the Indian population is necessary. Each population group in which the disease is prevalent has a different spectrum of β globin mutations and a few mutations account for most of the alleles. From 1997 to 2006, β-Globin gene mutation analysis was carried out in 1030 individuals. The common mutations in the population were screened by reverse dot blot (RDB). Deletion of 619 bp of the β globin gene was detected by gap-PCR. Rare mutations were screened using temporal temperature gel electrophoresis (TTGE) and DNA sequencing. Haplotyping of rare mutations was done by using established PCR-RFLP methods. Out of the 1030 individuals, 515 were homozygous or compound heterozygotes. Among the others, 178 had heterozygous β thalassaemia, 292 HbE-βthalassaemia, 27 HbS-β thalassaemia, 3 HPFH-β thalassaemia, 5 Lepore-β thalassaemia and 10 (δβ)0-β thalassaemia. Mutation analysis was performed on a total of 1545 β globin alleles from the subjects included in the study. The regional distribution of study subjects were: 533 from the South (Kerala, Tamilnadu, Andhra Pradesh and Karnataka), 529 from the East (Bihar, Orissa, West Bengal and other eastern states), 233 from the West (Gujarat, Maharashtra, Madhyapradesh and Rajasthan) and 92 from the Northern part of the country (Harayana, Punjab, Uttar Pradesh). A total of 41 mutations including 17 mutations that have not been reported earlier in this population were detected. Four, Codon 62/64 (−7bp), Codon 81/87 (−22bp), −25 (A→G) and IVS II- 613 (C→T) are novel mutations causing β thalassaemia. Thirteen mutations, which were detected in other populations but were not reported earlier in the Indian population, were identified. They included −29 (A→G), −87 (C→G), −90 (C→T), Codon 17 (A→T), Codon 22/23/24 (−17 bp), Codon 26 (G→T), Codon 36/37 (−T), Codon 41 (−C), IVS I-129 (A→C), IVS I-130 (G→C), IVS I-130 (G→A), Codon 106/107 (+G) and Codon 110 (T→C). Comparison of the spectrum of β thalassaemia mutations between different regions shows that there are considerable differences in the geographical distribution of mutations in the Indian population (Table). Haplotype analysis revealed that the rare mutations occured denovo and also through population migration. This data will aid in carrier detection, genetic counselling and prenatal diagnosis which play an important role in the prevention of the disease.Regional Distribution of β globin Mutations in India (%)MutationSouthEastNorthWestIVS I-5(G-C)57.4173.5154.3549.36CD 15(G-A)12.386.9903poly A (T-C)90.7300.86−28(A-G)4.5000IVS II-837 (T-G)2.81000CD 30(G-C)1.97.281.092.58CD 41/42(-TCTT)2.063.646.525.15619 bp DEL00.448.720.17IVS I-1(G-T)0.90.1511.9610.73CD 8/9 (+G)0.11.63.263OTHERS8.945.6714.135.15

Molecular identification of the microbiota of French sourdough using temporal temperature gradient gel electrophoresis

The microbiota of four industrial French sourdoughs (BF, GO, VB and RF) was characterized by PCR temporal temperature gel electrophoresis (TTGE). The TTGE technique reveals differences in the 16S rDNA V6–V8 regions of these bacteria. DNA was extracted directly from sourdough samples. A specific TTGE fingerprint was determined for 30 bacterial species, including members of the genera Lactobacillus, Leuconostoc and Weissella, all known to be present in sourdough. These sourdoughs contain different species of lactic acid bacteria (LAB) depending on ecological conditions prevailing in the different sourdough fermentations. Only a few LAB species were found to be competitive and became dominant. Lactobacillus sanfranciscensis was observed as the most frequently found species. In sourdough GO, L. sanfranciscensis, Lactobacillus panis and two new species, Lactobacillus nantensis and Lactobacillus hammesii, were detected. Sourdough BF contain L. sanfranciscensis, Lactobacillus spicheri and Lactobacillus pontis. In sourdough VB, which differed in the process temperature, we identified exclusively L. sanfranciscensis and Leuconostoc mesenteroïdes subsp. mesenteroïdes. Lactobacillus frumenti, L. hammesii and Lacobacillus paralimentarius became the predominant species in sourdough RF. Compared with conventional bacteriological methods, the use of this new molecular approach to analyze the sourdough ecosystem should therefore allow a more complete and rapid assessment of its specific microbiota.

Bacterial Community Structure Analyses to Assess Pollution of Water and Sediments in the Lake Shkodra/Skadar, Balkan Peninsula (8 pp)

Lake Skadar is the largest lake of the Balkan Peninsula, located along the Montenegro-Albanian border. The unique features of the lake and wide range of endemic, and rare or endangered plant and animal species, resulted in the classification of the Skadar as a wetland site of international significance. In spite of its importance, the lake is influenced by inflowing waters from the river Moraca and other regional rivers contaminated by the industry, municipal and agricultural activities in the area. Therefore, the lake has been the subject of various physical, chemical, biological and toxicological examinations. However, community-level analyses are most relevant to assess the effect of stressors on aquatic ecosystems. In the present study, bacterial community structure among differently polluted sites of the lake was compared using a genetic fingerprinting technique. Water and sediment samples were collected from five differently polluted sampling sites on the Lake Skadar in spring and autumn of the same year. The bacterial community structure in the samples was characterized and compared by temporal temperature gel electrophoresis (TTGE) analysis of polymerase chain reaction-amplified bacterial 16S rRNA genes. The TTGE analysis resulted in many distinguishable and reproducible band patterns, allowing a reliable comparison of bacterial communities among sampling sites. Results on the bacterial community structure revealed that three of the selected locations can be considered as sites that have not shown any pollution degradation determined by our method, due to similar structure of a bacterial community in the sediment samples. On the other hand, significant shifts in the bacterial community structure in the mouth of the rivers Moraca and Plavnica were shown. Since the results coincide with some of the bioassays and chemical analysis performed previously, the changes in bacterial community structure are explained as an effect of antropogenic pollution on the lake ecosystem by waters of the river Moraca and the stream Plavnica. The TTGE has proven to be an efficient and reliable method to monitor bacterial dynamics and community shifts in aquatic environments, especially in sediments. Within the variety of environmental quality assessments, the use of TTGE analyses from bacterial communities is strongly recommended, particularly as an initial investigation. However, in any conclusion on the state of the environment, the TTGE results should be combined to some other biological, chemical and hydrological data. Since prokaryotes are a crucial group of organisms in the biosphere, the ecosystem function studies are largely based on bacterial communities. Therefore, bacterial community structure analysis should be a part of an integrated weight of evidence approach in pollution assessment. In case of Triad approach, consisting of chemical analyses, bioassays, and community studies in the field, the TTGE bacterial community structure analyses should be placed in the later Triad leg. In comparison to other community studies, based on various biotic indices, the TTGE bacterial community analysis has proven to be very sensitive, reliable and less time consuming.

Evaluation of the arrayed primer extension resequencing assay for TP53mutation detection

Over the years we have screened for TP53 mutations in different patient materials using temporal temperature gel electrophoresis (TTGE) [1], followed by direct sequencing of samples with aberrant migrating bands to determine the nature of the sequence alteration. Mutations in the TP53 gene are associated with several different cancer types and have been shown to have both prognostic and predictive implications. In this project we are evaluating whether a commercial available array platform for sequencing the TP53 gene using a primer extension assay (APEX) is as sensitive, rapid and cost-effective as TTGE/sequencing. The array is designed by Asper Biotech [2]. Genomic DNA is amplified by PCR, and dUTP is incorporated. The amplification products are then concentrated and purified with spincolumns. Amplification products are fragmented by Uracil N-glycosylase, and unincorporated dNTPs are inactivated by shrimp alkaline phosphatase. The fragmented PCR products are mixed with thermosequenase and four fluorescence-labelled ddNTPs. The sample mixture is transferred to a chip that contains sequence-specific oligonucleotides. So far, exons 2–9 are included on the array. Genorama™ QuattroImager is used for scanning. The Genorama imaging system and genotyping software are used for imaging and semiautomatic sequence analysis. DNA samples from 48 primary breast carcinomas, 11 ovarian carcinomas and 34 cell lines were used for evaluation. Results from a titration experiment with different ratios of the Arg/Arg and Pro/Pro alleles on codon 72 in the TP53 gene showed that mutations could be detected even if the mutated cells were present in less than 5%. We have experienced that homozygous and hemizygous mutations occasionally are missed by the TTGE technique, but that they all were easily detected by APEX [3]. Detection of deletions and insertions, however, is not yet optimal using the APEX technology and they are frequently missed. For the tumour samples the resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strands. The strength of using the APEX technology is that both strands are simultaneously analyzed, and that no further sequencing is needed. It is rapid and sensitive. Cost-effectiveness is still under evaluation.

Raw cow milk bacterial population shifts attributable to refrigeration.

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.

DNA Based Classification of Food Associated Enterobacteriaceae Previously Identified by Biolog GN Microplates

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.

Distinct pattern of TP53 mutations in squamous cell carcinoma of the esophagus in Iran

Extremely high rates of squamous cell carcinoma of the esophagus (SCCE) are observed in Iran, reflecting unknown, genetic and/or epidemiological risk factors. Among genetic alterations in SCCE, TP53 mutations are the most frequent, vary among populations, and may provide clues on etiological mechanisms. We have analysed mutations in TP53 (exons 5-8) in 98 SCCE from Iran by temporal temperature gel electrophoresis and direct sequencing. We found 58 mutations in 49 patients (50%), with a high prevalence of C to T transitions at CpG dinucleotides (29.3%). The TP53 mutation pattern in Iran was significantly different from that observed in SCCEs from high incidence areas of China and Western Europe (P=0.007). Moreover, the prevalence of mutations at A : T base pairs (transitions and transversions) was higher in men than in women (38.7% vs 11.1%, P=0.033). COX-2 overexpression was detected in 69% of the cases evaluated (24/35), without significant association with TP53 mutation. Accumulation of nitrotyrosine, a marker of protein damage by excess levels of nitric oxide, was observed in tumor cells in six of 18 [corrected] cases analysed. These results are consistent with the hypothesis that several factors are involved in TP53 mutagenesis in Iran. These factors include a baseline of chronic inflammatory stress, which may have a multiplicative impact on the sensitivity of esophageal cells to exogenous factors of risk.

Germline PTEN mutations in Cowden syndrome-like families.

Cowden syndrome (CS) or multiple hamartoma syndrome (MIM 158350) is an autosomal dominant disorder with an increased risk for breast and thyroid carcinoma. The diagnosis of CS, as operationally defined...