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Aims: To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins. Methods and Results: A gene ( vhhP2) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24 V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non- V. harveyi species, including V. parahaemolyticus and V. alginolyticus. A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2. This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii, which is most closely related to V. harveyi. One of the V. campbellii strains was falsely identified as V. harveyi. Conclusions: vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non- V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi. However, this method can not distinguish some V. campbellii strains from V. harveyi. Significance and Impact of the Study: the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples. [ABSTRACT FROM AUTHOR]

The oral cavity has been proposed as a reservoir for H. pylori that could be responsible for the refractoriness of gastric infection to triple therapy (antibiotics, antimicrobials, and proton pump inhibitors). The aim of this study was to evaluate the efficiency of combined periodontal and triple therapy vs. triple therapy alone, in gastric H. pylori eradication in persons with H. pylori in the subgingival biofilm. Individuals positive for H. pylori in their gastric and oral samples, as determined by nested PCR, were treated either with periodontal and triple therapy or with triple therapy alone. Our results indicate that 77.3% of those treated with the combined therapy exhibited successful eradication of gastric H. pylori, compared with 47.6% who underwent only triple therapy. Analysis of these data suggests that periodontal treatment in combination with systemic therapy could be a promising approach to increasing the therapy's efficacy and decreasing the risk of infection recurrence. [ABSTRACT FROM AUTHOR]

Abstract: Molybdenum (Mo) nitrogenases consist of two components: dinitrogenase reductase (encoded by nifH) and the dinitrogenase or MoFe protein (encoded by nifDK). Nitrogenase enzyme of photosynthetic bacteria is responsible for hydrogen production. Therefore, primers were designed for the nitrogenase gene only. In this study, two primers (ND and NH) were designed after comparative genomic analysis of nifH and nifD gene sequences from public databases. The designed primers were used for the amplification of nifH and nifD genes to detect nitrogenase genes in photosynthetic bacteria. Initial detection was done using a monoplex Polymerase Chain Reactions (PCRs) followed by optimization of the PCR protocols. Subsequently, a duplex PCR was designed for amplification and detection of nifH and nifD genes in indigenous photosynthetic bacteria. Evaluation of the duplex PCR on six samples isolated from Palm Oil Mill Effluent (POME) showed that only four isolates contained both the nifH and nifD genes, indicating that these isolates were potential hydrogen-producing bacteria. PCR detection provides a rapid and efficient pre-identification of potential photosynthetic bacterial hydrogen producers. [Copyright &y& Elsevier]

Phomopsis azadirachtae Sateesh, Bhat & Devaki is the incitant of die-back disease of neem trees. Delayed appearance of conidia and presence of other microorganisms in the neem tissues are the obstacles in the rapid and accurate identification of P. azadirachtae. This work was carried out to develop a methodology for rapid detection of the pathogen in diseased tissues especially in the neem seeds. rDNA sequences of many Phomopsis spp. were retrieved from the database and were subjected for multiple alignment to select a 179 bp conserved sequence. This was used to design Phomopsis specific primer pair (Forward and Reverse) having the potential to produce a 154 bp product in PCR. The primer pair was utilised to detect the presence of P. azadirachtae in diseased neem seeds and other tissues. This is the first report on the PCR-based detection of P. azadirachtae directly in die-back diseased neem tissues. This method can be employed for rapid and reliable detection of P. azadirachtae in die-back affected neem seeds. Hence it will have very good application in seed health testing laboratories. [ABSTRACT FROM AUTHOR]

Introduction and purpose: Yersinia enterocolitica belongs to the family of Enterobacteriaceae and is a psychrophilic pathogen that is associated with foodborne infections. It usually causes gastroenteritis, mesenteric lymphadenitis, and septicemia. This study aimed to molecular detection, biotyping, and serotyping of Yersinia enterocolitica isolated from chicken livers in Tabriz. Materials and Methods: One hundred chicken liver samples were collected randomly from poultry slaughterhouses in Tabriz for three months. After enrichment process, the presence of Yersinia enterocolitica in studied samples was determined through culture-based methods, biochemical and molecular tests. Then the biotype and serotype of the isolates were determined. Results: 31 samples (31%) were positive for Yersinia enterocolitica by both phenotypic and molecular assays. Among positive samples, 25 (80.64%) had non-pathogenic biotype 1 A with serotype O: 5 (23 samples) and O: 8 (2 samples). 6 (19.36%) had biotype 1B and all of them had O: 3 serotype. The serotype Yersinia enterocolitica O: 9 was not found. Conclusion: the present study highlighted the significance of chicken liver as potential source of Yersinia enterocolitica infection in Tabriz city. • Chicken liver may act as potential sources of Yersinia enterocolitica infection in Tabriz city. • Most of the Yersinia enterocolitica isolates belonged to non-pathogenic biotype 1 A. • Most of the Yersinia enterocolitica isolates belonged to serotype O: 5. [ABSTRACT FROM AUTHOR]

Meru, Kenya has watersheds which are shared by wildlife, humans and domesticated animals. These surface waters can be contaminated by the waterborne pathogen Cryptosporidium. To quantify the seasonality and prevalence of Cryptosporidium in Meru regional surface waters, we used a calcium carbonate flocculation (CCF) and sucrose floatation method, and a filtration and immunomagnetic bead separation method, each of which used PCR for Cryptosporidium detection and genotyping. Monthly water samples were collected from January through June in 2003 and 2004, bracketing two April-May rainy seasons. We detected significant seasonality with 8 of 9 positive samples from May and June ( p < 0.0014), which followed peak rainy season precipitation and includes some of the subsequent dry season. Six of 9 positive samples revealed C. parvum, and 3 contained C. andersoni. None contained C. hominis. Our results indicate that Meru surface waters are Cryptosporidium-contaminated at the end of rainy seasons, consistent with the timing of human infections reported by others from East Africa and contrasting with the onset of rainy season peak incidence reported from West Africa. [ABSTRACT FROM AUTHOR]

Rhizobia, producing species-specific exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identification and subsequent discrimination between Rhizobium species has been developed and tested. For identification of R. leguminosarum, primer sets I–III complementary to sequences of rosR, pssA and pssY genes were proposed. Further sets of primers (IV–VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a wide range of R. leguminosarum strains isolated from different host plants nodules originating from different regions of Poland. We demonstrate a high discriminating power of primer sets I–III that allow distinguishing R. leguminosarum and two closely related species, R. etli and R. gallicum. This new approach is applicable to identification of R. leguminosarum strains, originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA might be useful markers to differentiate and define relationships within a group of R. leguminosarum strains. [ABSTRACT FROM AUTHOR]

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of São Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fDl/rPl was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known. [ABSTRACT FROM AUTHOR]

1 Populations of an indigenous longhorn beetle, the red oak borer Enaphalodes rufulus (Haldeman) (Coleoptera: Cerambycidae), recently reached epidemic levels in the Ozark National Forests of Arkansas and Missouri, resulting in extensive tree mortality. 2 The factors regulating E. rufulus populations are largely unknown. Ants appear to be the most abundant potential predators in the Ozarks and have been shown to play a role in regulating populations of other forest insects. 3 The main objective of the present study was to determine whether ants are predators of early E. rufulus life stages by direct observation of E. rufulus eggs artificially placed on tree trunks and by development of molecular tools to detect E. rufulus DNA within ant gut contents. 4 Three hundred and eighty E. rufulus eggs were applied to ten northern red oaks. Ants ate or carried away 72% of the eggs within 1 h. Camponotus pennsylvanicus (De Geer) and Aphaenogaster tennesseensis (Mayr) were identified as the two primary ant species observed in the study. 5 A portion of the mitochondrial 16S rRNA gene of E. rufulus was sequenced and polymerase chain reaction primers were developed to detect E. rufulus DNA in the guts of C. pennsylvanicus. Enaphalodes rufulus DNA persisted in ant gut contents for at least 24 h after ingestion under laboratory conditions, and E. rufulus DNA was detected in field-collected ant populations, suggesting the natural occurrence of ant predation on this insect. [ABSTRACT FROM AUTHOR]

Abstract: Bacteriophage infections of starter lactic acid bacteria (LAB) pose a serious risk to the dairy industry. Nowadays, the expanding use of valuable Lactobacillus strains as probiotic starters determines an increase in the frequency of specific bacteriophage infections in dairy plants. This work describes a simple and rapid Polymerase Chain Reaction (PCR) method that detects and identifies bacteriophages infecting Lactobacillus casei/paracasei, the main bacterial species used as probiotic. Based on a highly conserved region of the NTP-binding genes belonging to the replication module of L. casei phages φA2 and φAT3 (the only two whose genomes are completely sequenced), a pair of primers was designed to generate a specific fragment. Furthermore, this PCR detection method proved to be a useful tool for monitoring and identifying L. casei/paracasei phages in industrial samples since specific PCR signals were obtained from phage contaminated milk (detection limit: 104 PFU/mL milk) and other commercial samples (fermented milks and cheese whey) that include L. casei/paracasei as probiotic starter (detection limit: 106 PFU/mL fermented milk). Since this method can detect the above phages in industrial samples and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms, or processes which involve phage-deactivating conditions. [Copyright &y& Elsevier]

Aims: Wheat stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici ( Pst) , is the most important foliar disease on wheat in China. Early molecular diagnosis and detection of stripe rust will provide a useful aid to the accurate forecast and seasonal control of this destructive disease. Our objective was to develop PCR assays for the rapid identification and detection of P. striiformis. Methods and Results: The genomic DNA of P. striiformis and P. triticina were amplified by a pair of primers derived from conserved β-tubulin gene sequence. A 235-bp specific DNA fragment of P. striiformis was isolated and purified. Based on its sequence, another two primer sets were designed successfully to obtain new sequence-characterized amplified region (SCAR) markers of P. striiformis, which could be amplified in all test isolates of P. striiformis, whereas no DNA fragment was obtained in other nontarget wheat pathogens. The detection limit of the primer set YR (f)/YR (r1) was 2·20 pg μl−1. The new SCAR markers of P. striiformis can also be detected in Pst-infected wheat leaves postinoculated for 2 days. Conclusions: Our assays are significantly faster than the conventional methods used in the identification of P. striiformis. Significance and Impact of the Study: Development of a simple, high-throughput assay kit for the rapid diagnosis and detection of wheat stripe rust would be anticipated in a further study. [ABSTRACT FROM AUTHOR]

Abstract: One hundred eighteen samples (21 species) of wild marine invertebrates were collected from western and southern coastal area of Korean Peninsula. Four of 78 (18 species) samples collected at Namhae (South) area were positive for nodavirus in nested PCR test. Of the 40 samples (5 species) collected at Hwanghae (West) areas, all samples were negative for nodavirus in both RT-PCR and nested PCR tests. Positive nested PCR results were obtained from the following species: Charybdis bimaculata Charybdid crab; Pandalus hypsinotus Southern humpback shrimp and Mytilus galloprovincialis Mediterranean mussel. Phylogenetic analysis based on the partial nucleotide sequence (177 bases) of the RNA2 coat protein gene showed that the four strains were highly homologous (100%) and closely related to that of the known betanodaviruses, redspotted grouper nervous necrosis virus (RGNNV). These results indicate that nodavirus is present from wild marine invertebrates in the southern coastal areas of Korean Peninsula. These subclinically infected marine invertebrates may constitute an inoculum source for betanodavirus infection and cause mortality in cultured fishes in Korea. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Staphylococci harbouring antibiotic resistance (AR) genes may represent a hazard for human health and, as other resistant food-related bacteria, they contribute to the spread of AR. In this study, we isolated resistant staphylococci from an entire swine production chain and investigated the occurrence of 11 genes [aac(6′)Ie-aph(2″)Ia, blaZ, mecA, vanA, vanB, ermA, ermB, ermC, tet(M), tet(O) and tet(K)] encoding resistance to some antibiotics largely used in clinical practice. The 66 resistant staphylococcal isolates were identified as Staphylococcus epidermidis (27 isolates), Staphylococcus aureus (12), Staphylococcus xylosus (12), Staphylococcus simulans (5), Staphylococcus pasteuri (4), Staphylococcus carnosus (3), Staphylococcus lentus (2) and Staphylococcus sciuri (1). Specific-PCR detection of AR genes showed the prevalence of the tet(K) gene in most of the isolates (89.4%), followed by tet(M) and ermC (about 75%); mecA was detected in more than half of S. aureus and S. epidermidis isolates. The genes vanA and vanB were not retrieved. It was found that a high proportion of coagulase-positive and -negative isolates are multidrug-resistant and some of them carry up to six AR genes. Our findings show that the swine production chain is a source of antibiotic-resistant staphylococci suggesting the importance of resistance surveillance in the food production environment. [Copyright &y& Elsevier]

A protist tentatively designated “ Pseudoperkinsus tapetis” belonging to the eukaryotic group Ichthyosporea (Mesomycetozoa) was previously isolated from carpet shell clams in Galicia (northwest Spain). In the present study, based on molecular data, a potential P. tapetis specimen was identified from the gill tissues of the mussel Adipicola pacifica associated with whale carcasses (generating chemosynthetic-based ecosystems) collected at shelf depths in the northwest Pacific (southwest Japan). Small subunit ribosomal DNA sequences (1751 sites) of the genotypes of P. tapetis from Spain and Japan were almost identical (only one substitution and one insertion/deletion difference). On the other hand, differences of 10 and 8 substitutions were found in two internal transcribed spacer regions of ribosomal DNA, ITS1 (288 sites) and ITS2 (251 sites) between these two genotypes, respectively, indicating that they are genetically different at the population level. These findings suggest that P. tapetis occurs worldwide and can associate with (and possibly infect) various types of bivalves. Further, a PCR method to specifically detect the P. tapetis cells in the host was also established. [ABSTRACT FROM AUTHOR]

Despite the high sensitivity and specificity of PCR for infectious disease diagnostics, it has presented low sensitivity for Mycobacterium leprae DNA detection in the tuberculoid pole (TT and BT) of leprosy. In order to demonstrate the effect of amplicon size on the efficacy of PCR detection of M. leprae DNA in skin lesions of leprosy patients, two pairs of primers targeting the M. leprae genomic DNA, RLEP3 (X17153), were used to amplify fragments of 372 and 130-bp until their PCR end-points were reached after 40 reaction cycles. Skin biopsies of leprosy lesions in 110 non-treated patients were used for bacilloscopy index (BI) analysis and PCR tests. The 130-bp fragment was detected in 73.6% of samples (81/110), and classified as TT (40%), BT (55.5%), and 100% of BB, BL and LL. The 372-bp fragment was detected in 52.7% and classified as TT (13.3%), BT (33.3%), BB (64.7%), BL (83.3%), and LL (95.2%). The BI of biopsies was positive in 39.1% of samples, classified as TT (0%), BT (2.2%), BB (64.7%), BL (91.6%), and LL (95.2%). The shorter amplicon (130-bp) has improved diagnosis by 20.9 and 34.5% in relation to the 372-bp fragment and the BI, respectively, and has shown a superior sensitivity (73.6%), specificity (100%) and accuracy (86.2%). The 130-bp amplicon could not detect % of positive BI of biopsies in BT cases. Therefore, for confirmatory diagnosis, we propose the use of PCR detection of the 130-bp genomic target, especially when the tuberculoid pole forms are considered, which has reached 51.6% of positivity in this group. [ABSTRACT FROM AUTHOR]

Bacillus species are well known for their ability to control plant diseases through various mechanisms, including the production of secondary metabolites. Bacillus subtilis DFH08, an antagonist of Fusarium graminearum, and other Bacillus spp. that are antagonists of common fungal pathogens of canola were screened for peptide synthetase biosynthetic genes of fengycin and bacillomycin D. Specific polymerase chain reaction (PCR) primers identified B. subtilis strains DFH08 and 49 for the presence of the fenD gene of the fengycin operon. Bacillus cereus DFE4, Bacillus amyloliquefaciens strains DFE16 and BS6, and B. subtilis 49 were identified for the presence of the bamC gene of the bacillomycin D synthetase biosynthetic operon. Both fengycin and bacillomycin D were detected in the culture extract of strain Bs49, characterized through MALDI–TOF–MS (matrix-assisted laser desorption ionization – time of flight – mass spectrometry), and their antifungal activities demonstrated against F. graminearum and Sclerotinia sclerotiorum. This study designed and used specific PCR primers for the detection of potential fengycin- and bacillomycin D-producing bacterial antagonists and confirmed the molecular detection with the biochemical detection of the corresponding antibiotic produced. This is also the first report of a B. cereus strain (DFE4) to have bacillomycin D biosynthetic genes. Bacteria that synthesize these lipopeptides could act as natural genetic sources for genetic engineering of the peptide synthetases for production of novel peptides. [ABSTRACT FROM AUTHOR]

Citrus greening or Huanglongbing disease caused by a nonculturable, phloem-limited bacterium, Candidatus Liberibacter asiaticus, is one of the most serious and destructive citrus diseases in the world. The presence of this disease in citrus orchards in the North-Eastern region of India has been confirmed through polymerase chain reaction (PCR)-based molecular detection. Samples of DNA extracted from leaves of putatively infected different citrus cultivars and from the vector of the disease, Diaphorina citri, were subjected to analysis using PCR and produced DNA amplicons characteristic of Ca. L. asiaticus. This is the first molecular evidence confirming the presence of greening disease and its psyllid vector in NE India. [ABSTRACT FROM AUTHOR]

Salmonella spp. bacteria are responsible for some of the most important zoonoses worldwide. Because Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) has been recently reported to be an experimental vector of Salmonella Enteritidis, it would be of benefit to evaluate the presence of this bacterium in mites. A molecular detection tool associating a simple filter-based DNA preparation with a specific 16S rDNA Salmonella sp. polymerase chain reaction (PCR) amplification was described. The limit of detection with this method was 2 × 104 bacteria per mite. To adapt this technique for large-scale studies, two sizes of mite pools were tested and a preliminary investigation was carried out on mites from 16 currently or previously contaminated farms. Mites sampled from one farm of each type were positive for Salmonella, suggesting that Dermanyssus could act as a reservoir between flocks. In further investigations, it will be necessary to carry out a large-scale study to assess the role of D. gallinae in the epidemiology of avian salmonellosis. [ABSTRACT FROM AUTHOR]

Abstract: Because the T-cell receptor gamma (TCRγ) gene is rearranged at an early stage of T-cell development in both TCRαβ and TCRγδ lineages, it has been preferentially targeted to detect T-cell clonality in human lymphoma/leukemia. We isolated 22 independent cDNA clones encoding canine TCRγ and the following analysis of nucleotide sequences using the dog genome database revealed that the canine TCRγ locus contains at least four repertories of variable genes that can be organized into two distinct subgroups and six repertories of joining genes belonging to two distinct subgroups according to the nucleotide sequence similarity. The findings allowed us to design PCR primers that were directed to the conserved or specific nucleotide sequences for each subgroup of variable and joining genes. By using four different combinations of primers, a PCR-based analysis was performed on cell samples collected from T-cell lymphoma/leukemia and B-cell lymphoma cases and hyperplastic and normal lymph nodes. All cell samples from 11 T-cell malignancy cases exhibited clonal amplification by two out of four primer combinations. This finding was considered to be valuable in PCR-based analysis for detecting T-cell clonality in canine lymphoma/leukemia. [Copyright &y& Elsevier]

Aim: To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. Methods and Results: A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, ‘morcilla’ and ‘fiambre de magro adobado’ obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. Conclusions: Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. Significance and Impact of the Study: Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR). [ABSTRACT FROM AUTHOR]

A reliable and sensitive PCR assay to specifically detect trichothecene-producing Fusarium spp. in milling fractions and kernel tissue of naturally infected durum wheat is reported. Assays were based on a combination of primers derived from the trichodiene synthase and the β-tubulin genes. The occurrence of toxigenic Fusarium spp. in semolina and wheat tissue (grain ends, crease, pericarp, aleurone layer, germ and albumen) was detected, even for a weakly contaminated wheat sample. Penetration of toxigenic Fusarium spp. into the interior of durum wheat kernel was demonstrated for the Nefer variety, indicating that none of the tissue structures within the wheat kernel acted as an effective barrier to fungal invasion. Moreover, after inoculation by toxigenic Fusarium strains, semolina was shown to allow high yields of trichothecenes, while bran was demonstrated to contain biochemical inhibitors able to significantly reduce trichothecene production. These results will be useful in improving breeding strategies to control trichothecene contamination of durum wheat kernels. [ABSTRACT FROM AUTHOR]

A sensitive and specific assay was developed to detect potato blackleg caused by Pectobacterium atrosepticum in potato. Primers PEAF and PEAR from the Rhs family gene homologous to RhsA (cell envelope biogenesis – outer membrane) were used to amplify a 904 bp DNA fragment. PCR was used to detect the pathogen in artificially inoculated potato seed tubers. The PCR product was only produced from 12 isolates of P. atrosepticum from various countries among 36 isolates of other species of Pectobacterium, Pseudomonas, Xanthomonas, as well as Escherichia coli and the soilborne fungus Fusarium oxysporum f.sp. dianthi. [ABSTRACT FROM AUTHOR]

Aim: To develop a diagnostic assay based on polymerase chain reaction for the detection of Magnaporthe grisea from infested rice seeds Methods and Results: Primers were designed based on the nucleotide sequence of the mif 23, an infection-specific gene of M. grisea. The primers amplified target DNA from genetically and geographically diverse isolates of the pathogen. The lowest concentration of template DNA that led to amplification was 20 ρg. No PCR product was detected when DNA from other fungi was used, indicating the specificity of the primers. With this PCR based seed assay, M. grisea was detected in rice seedlots with infestation rates as low as 0.2%. Conclusion: The PCR detection of M. grisea is simple, rapid, specific, sensitive and suitable for the routine detection of the pathogen in infested seeds. Significance and Impact of the Study: Introduction of the blast fungus into new areas where it has not been previously recorded could be avoided by the detection of infested seedlots. A PCR-based seed assay could facilitate risk assessment of naturally infested rice seeds; help design management programs and optimize fungicide use. [ABSTRACT FROM AUTHOR]

A PCR-based heteroduplex assay was used to determine the presence and composition of Bacillus species in 11,059 Environmental Protection Agency PM2.5 aerosol samples from 11 U.S. cities. The assay differentiated three groups: Type A containing Bacillus anthracis and very closely related, often pathogenic, Bacillus cereus and Bacillus thuringiensis strains; Type B containing other B. cereus and B. thuringiensis strains; and a third group of more-distantly related Bacillus species. Eight of the 11 cities were positive for Bacillus species in 50% or more of the samples, and the percent of aerosol samples that contained the HD Type A group ranged from 3% to 32%. Cities from the eastern half of the United States generally contained a higher frequency and broader diversity of Bacillus species than the western half of the United States. Positive samples were detected throughout the year. These results have implications for pathogen detection in environmental samples, understanding the natural evolution of new pathogenic strains, and incidence of infection caused by strains of the B. cereus subgroup. [ABSTRACT FROM AUTHOR]

Aims: A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out. Methods and Results: The technique used previously developed nanoparticles modified with a 21-mer oligonucleotide. This sequence, sharing homology with all the L. monocytogenes strains, was selected on hlyA gene and located outside the desired specific PCR site to avoid cross-contaminations. Capture probe properties, in term of spacer length and purification, were determined to obtain the highest hybridization efficiency. Its specificity was tested in hybridization experiments with nontarget bacterial species. Any inhibitory effect of the nanoparticles on PCR was also examined. The amplification performed with the purified DNA could reliably identify a 10 CFU ml−1 contamination rate. Conclusions: The optimized purification method showed a high specificity and sensitivity, with a detection level one log more sensitive than PCR carried out with nucleic acids obtained using commercial nanoparticles. Significance and Impact of the Study: The method, avoiding pre-enrichment, provides a rapid alternative to conventional microbiological detection methods. Furthermore, it is suitable for automation and can be proposed for the screening of a large number of samples. [ABSTRACT FROM AUTHOR]

Aims: To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. Methods and Results: Specificity of nuc targeted primers (Pri1–Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 103 CFU g−1. The two RTQ-PCR systems, incorporating SYBR-Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93·3%). Conclusions: PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food. Significance and Impact of the Study: The SYBR-Green I real-time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost. [ABSTRACT FROM AUTHOR]

Abstract: Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for meta-cleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 103–104 or 105–106 gene copies g−1 soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination. [Copyright &y& Elsevier]

tmoA and related genes encode the α-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence. [ABSTRACT FROM AUTHOR]

A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (TcR) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of TcR streptomycetes originated from bulk and rhizosphere soil. Fewer TcR streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene–carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which TcR streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated the influence on longevity and fecundity of Flavescence dorée phytoplasma (FDP), the agent of a grapevine yellows disease, in the experimental vector Euscelidius variegatus Kirschbaum. Late instar nymphs were exposed to one or the other of two strains of FDP (FD92 and FD2000) by feeding on infected broad bean (Vicia faba L.) or on healthy broad bean or maize (Zea mays L.) for an acquisition access period of 13 days. Detection of FDP in individual insects was done with PCR assays and revealed that almost all exposed leafhoppers had acquired FDP, for both FD92 and FD2000 strains. FDP infection significantly reduced the life span of males and females (ANOVA of the quartiles of survival distribution and Weibull scale parameter). FDP-exposed females produced significantly fewer nymphs. The two FDP strains had similar effects on reduction of survival and fecundity of leafhoppers. There was no significant differences in longevity of E. variegatus males exposed to FD broad bean than held on healthy broad bean or maize, but female survival and fecundity were reduced when they fed on maize versus healthy broad bean. [Copyright &y& Elsevier]

Abstract: PVYN-W is one of the variant populations of Potato virus Y (PVY). This variant is of concern in seed potato production and requires a specific diagnosis since it induces more or less symptomless infections and is not detectable easily in field inspections. Moreover, this variant is serologically indistinguishable from the common strain PVYO. This study describes a simple and specific molecular detection test for the PVYN-W variant using a PCR protocol based on the recombinant point within the HC-Pro/P3 region of PVYN variants (PVYNTN, PVYN-W). To avoid both detection of recombinant PVYNTN and PVYN-W isolates, a forward PVYN-like primer located in the HC-Pro region coupled to a reverse PVYO-like primer located in the NIa region was designed to amplify a specific PCR product of 4114 nt from PVYN-W isolates. This technique was assessed on 41 PVY reference and field isolates. Only isolates referenced as PVYN-W were amplified and gave the expected PCR product of 4114 nt, whereas no band was obtained from PVYN, PVYNTN or PVYO isolates. In conclusion, this PVYN-W diagnosis tool is rapid, easy-to-use and suitable for large-scale testing in laboratories of seed potato certification. [Copyright &y& Elsevier]

Specific polymerase chain reaction (PCR) primers were developed from amplified fragment length polymorphism (AFLP) fragments ofPyrenophora teres, the causal agent of net blotch on barley leaves. The primers were designed specifically to amplify DNA fromP. teresf.teres(net form) and allow its differentiation fromP. teresf.maculata(spot form), which is morphologically very similar toP. teresf.teresin culture. The PCR amplification was carried out successfully from DNA extracted from fungal mycelium. The PCR assay was validated with 60 samples ofPyrenophoraspecies. The amplification with four designed PCR primer pairs providedP. teresform-specific products. No cross-reaction was observed with DNA of several other species, such asP. tritici-repentis,P. gramineaandHelminthosporium sativum. [ABSTRACT FROM AUTHOR]

A collection ofPseudomonas corrugataandP. mediterraneastrains, two closely related species, was evaluated for the presence and variability ofphaloci. Using PCR methods that specifically amplify segments of medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) synthase genes, we demonstrated the presence ofphaC1andphaC2in allP. mediterraneastrains tested and in six out of 56 strains ofP. corrugatascreened. The remaining 50 strains ofP. corrugatayielded only thephaC2subgene fragment on detection by a combined PCR-restriction endonuclease analysis method or a semi-nested PCR-amplification approach. A Southern hybridization study on a representative strain from this group, however, indicated the presence of thephaC1gene. Nucleic acid sequences of the subgenephaCfragments of the representative strains from the three groups showed an overall similarity ranging from 95% to 100%. The major repeat-unit monomers of the mcl-PHAs isolated from these selected strains are ß-hydroxyoctanoate (33-47 mol%) and ß-hydroxydecanoate (26-36 mol%). These results differentiate for the first time the strains ofP. corrugatainto twopha-distinguishable groups. This study also documents for the first time the production of mcl-PHA inP. mediterranea. [ABSTRACT FROM AUTHOR]

Abstract: Some lactic acid bacteria can induce viscosity in wine, beer and cider by production of exopolysaccharides (EPS). A polymerase chain reaction (PCR) assay was previously described for the detection of ropy Pediococcus damnosus strains in wine [J. Appl. Microbiol. 90 (2001) 535]. The primers used in that study, PF5 and PF6, are investigated in addition to new primers which broaden the range of spoiling agents detectable by PCR. Primers PF1 and PF8 allow the amplification of DNA from ropy P. damnosus strains isolated from wine, as was observed with PF5 and PF6. In addition, PF1 and PF8, unlike PF5 and PF6, are able to generate an amplicon using template DNA from a ropy P. damnosus strain isolated from cider and a ropy Oenococcus oeni strain isolated from champagne. Two different ropy Lactobacillus species were also isolated, but their DNA was not amplified using primers PF1 and PF8. The new primers PF1 and PF8 were chosen from the sequence of gene dps, a putative glucan synthase gene, found across all the ropy P. damnosus strains isolated, from both wine or cider, and also in a ropy O. oeni strain. To our knowledge, this is the first time that an EPS-producing O. oeni strain is described. Glucan biosynthesis was assessed by agglutination tests done with Streptococcus pneumoniae type 37-specific antibodies, which specifically detect glucan-producing cells. The results show that there is a direct correlation between glucan production and detection of gene dps. Therefore, Dps is considered a candidate for the glucan synthase enzyme responsible for EPS production by ropy strains of P. damnosus and O. oeni. [Copyright &y& Elsevier]

Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected inRalstonia solanacearumrace 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165?bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125?bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of otherR. solanacearumstrains or of other related bacteria. PCR detection limit for the pathogen was 2 × 102?cfu. [ABSTRACT FROM AUTHOR]

c.l. watson, r.j. owen, b. said, s. lai, j.v. lee, s. surman-lee and g. nichols. 2004.To investigate treated water distribution systems in England as a source ofHelicobacter pylori.Water and biofilms were obtained from 11 domestic and seven educational properties and from hydrants, reservoirs and water meters supplied by three water utilities. Samples were cultured on nonselective and antibiotic containing media combined with immunomagnetic separation concentration. Viable helicobacters were not detected in any of the 151 samples butHelicobacter-specific PCR assays detected DNA in 26% of samples from domestic properties, schools and hydrants with the highest frequency in biofilms (42%). Direct sequencing of six selected amplicons confirmed>95% sequence homology toH. pylori.While viable helicobacters were not isolated, evidence was obtained for the presence ofHelicobacterDNA, including that ofH. pylori. Biofilms on surfaces within water distribution systems may act either as sites for the passive accumulation of helicobacters or as potentially important reservoirs of infection.Our findings strengthen evidence thatH. pylorimay be transmitted through drinking water. However, there is currently no evidence that viable cells can survive the disinfection levels used in UK mains supplies and the health risk from this source remains unclear. [ABSTRACT FROM AUTHOR]

Purified DNA from isolates of Fusarium poae, Fusarium sporotrichioides, Fusarium kyushuense and Fusarium langsethiae was used as a template to amplify a 658-bp fragment from the trichodiene synthase (tri5) gene of these fungi with the gene-specific PCR primer pair Tox5-1/Tox5-2. Fragments obtained were isolated and sequenced. DNA sequence alignments revealed high similarity between the sequences derived from F. sporotrichioides and F. langsethiae (98.7%) and less similarity between the latter species and F. poae (90.9%). Phylogenetic analysis of the aligned sequences using the tri5 sequence of Fusarium pseudograminearum as an outgroup revealed clear separation between one group consisting of F. poae and F. kyushuense and another consisting of F. sporotrichioides and F. langsethiae. The two latter species could not be distinguished phylogenetically on the basis of their tri5 sequences.Taxon-specific reverse primers were designed from the aligned sequences and combined with the tri5 gene-specific forward primer Tox5-1. The new reverse primers enabled specific amplification of a fragment of approximately 400 bp from DNA isolated from F. sporotrichioides, F. poae, F. langsethiae and F. kyushuense, respectively. All primers were tested for cross-reactivity with DNA from 26 fungal species potentially capable of producing trichothecenes. Only the primer designed for F. langsethiae cross-reacted with F. sporotrichioides. PCR assays were applied in analysis of artificially and naturally infected samples of barley and oats. On artificially infected barley, species were selectively detected by the corresponding primers. In naturally infected oats, F. langsethiae was identified by the combination of two PCR assays designed for detection of F. sporotrichioides and F. langsethiae, respectively. [Copyright &y& Elsevier]

A total of 78 raw and 123 processed and ready-to-eat retail food samples were used to assess the presence of motile Aeromonas spp. harboring virulence genes (cytotoxic enterotoxin and hemolysin genes) using a recently described PCR method in comparison with the conventional cultivation method based on the use of Ampicillin-Dextrin Agar (ADA) medium. With the ADA-based method, 65/201 (32.3%) samples showed presumptive Aeromonas spp. colonies whereas the PCR method revealed the presence of Aeromonas spp. harboring the targeted virulence genes in 51/201 (25.4%) of the tested samples. The rate of contaminated samples and the presence of pathogenic Aeromonas were significantly lower with both methods for processed than in case of raw samples. A polyphasic identification approach including biochemical and molecular techniques was applied to a selection of 34 PCR-positive presumptive Aeromonas isolates. Following fatty acid methyl ester (FAME) analysis and amplified fragment length polymorphism (AFLP) fingerprinting, a total of 33 isolates (97%) could be identified to the DNA hybridization group (HG) level. The majority of these isolates belonged to the species Aeromonas hydrophila HG3 (50%) and Aeromonas veronii biovar sobria (HG8/10) (38%). Molecular characterization of PCR amplicons obtained from these strains by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) fingerprinting and PCR-Amplicon Sequence Analysis (PCR-ASA) allowed classification of all strains in a known PCR-RFLP and PCR-ASA type. In conclusion, the current findings demonstrate that the combined use of PCR-based virulence marker detection, PCR-RFLP and PCR-ASA offers a rapid, sensitive, and specific system to assess the presence and prevalence of Aeromonas spp. harboring virulence markers in food samples. [Copyright &y& Elsevier]

Screening for and detection of the novel extended spectrum β-lactamase (ESBL), GES-2 produced by Pseudomonas aeruginosa remains a problem in the clinical microbiology laboratory. This study aimed to compare the normally used ESBL screening agent ceftazidime, with molecular detection, to demonstrate the presence of GES-2 ESBL production in clinical isolates of P. aeruginosa. Ceftazidime was found unreliable as an ESBL screening agent, with a specificity of 34.4%, when National Committee for Clinical Laboratory Standards resistance criteria for P. aeruginosa were employed. An improved PCR detection method was devised, that amplified a 371 bp segment of blaGES-2. This should lead to more cost-effective DNA sequencing and sequence interpretation in the laboratory. [Copyright &y& Elsevier]

m.n. romanov, r.v. bato, m.t. yokoyama and s.r. rust. 2004. The aims of this study were to develop a sensitive and more rapid detection of Propionibacterium acidipropionici DH42 in silage and rumen fluid samples, and to explore its 16S rRNA sequence-based phylogeny. Nested polymerase chain reaction (PCR) was used with DH42-specific primers dhb1 and dhb2 for the secondary amplification of a 1267-bp fragment of 16S rRNA encoding gene. Using the established protocols for PCR amplification, as low as 102 and 103 CFU ml−1 of strain DH42 in silage extracts and rumen fluid, respectively, were detected. To determine phylogenetic relationships between DH42 and other representatives of Propionibacterineae, a 1529-bp fragment of its 16S rRNA was amplified by PCR and sequenced. The propionibacterium DH42 formed a cluster with Eubacterium combesii, P. acidipropionici and P. microaerophilus. 16S rRNA-based PCR detection technique was developed for DH42 in silage and rumen fluid samples. The 16S rRNA sequence confirmed the earlier identification of strain DH42 as P. acidipropionici. However, variable nucleotide positions were revealed. Variability of 16S rRNA sequence within the species P. acidipropionici, determined in this study, poses the need of re-sequencing for some species of the suborder Propionibacterineae for a more reliable classification. [ABSTRACT FROM AUTHOR]

We report on the development of a PCR-based assay to detect Pasteuria penetrans infection of Meloidogyne arenaria in planta using specific primers for recently sequenced sigE, spoIIAB and atpF genes of P. penetrans biotype P20. Amplification of these genes in crude DNA extracts of ground tomato root galls using real-time kinetic PCR distinguished infected from uninfected M. arenaria race 1 by analysis of consensus thresholds for single copy genes. Fluorescent in situ hybridization (FISH) using the sigE primer sequence as a probe shows hybridization to P. penetrans cells in various stages of vegetative (pre-endospore) development. Ratios of gene copies for sigE and 16S rDNA were obtained for P. penetrans and compared to Bacillus subtilis as a genomic paradigm of endospore-forming bacteria. Phylogenetic analysis of the sigE gene from Gram-positive, endospore-forming bacteria finds P. penetrans most closely related Paenbacillus polymyxa. The sporulation genes (spo genes), particularly sigE, have sequence diversity that recommends them for species and biotype differentiation of the numerous Pasteuria isolates that infect a large number of plant-parasitic nematodes. [Copyright &y& Elsevier]

A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300–600%, when the etch depth is varied from 20 to 50 μm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 μl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step. [Copyright &y& Elsevier]

A polymerase chain reaction method to detect soil-borne plant pathogens such as Pythium ultimum, Plasmodiophora brassicae, and Verticillium dahliae in soil was developed and used with a range of soil textures. DNA extraction was based on lyses with alkali buffer (pH 9.0) containing sodium dodecyl sulfate, extraction with benzyl chloride, and collection by ethanol precipitation. Four modified protocols – purification of extracted DNA from soil, independent addition of glass beads or skim milk to the extraction buffer, and addition of bovine serum albumin (BSA) to the PCR reaction mixture – were evaluated to enhance the sensitivity of detection. The effectiveness varied for three tested pathogens, but none of the four protocols negatively affected PCR detection. DNA purification was essential for detecting the three tested pathogens. Physical disruption with glass beads and the addition of BSA to the PCR reaction mixture were necessary for detecting V. dahliae, and the addition of skim milk was needed for Pl. brassicae. Additions of BSA and skim milk enhanced the detection of P. ultimum. The developed protocol seems applicable to the range of soil textures that are naturally inhabited by these three pathogens. By integrating multiple protocols to enhance sensitivity, PCR can be used to detect various soil microorganisms. [ABSTRACT FROM AUTHOR]

Streptococcus thermophilus, Bifidobacterium infantis Y1 and Bifidobacterium breve Y8 strains were identified and enumerated by PCR assay in human fecal samples after intake of the pharmaceutical preparation VSL-3 or yogurt. ThI/ThII primer set, specific for S. thermophilus, was selected testing its specificity against several strains of enterococci, streptococci and other genera colonizing the human intestine. A culture-independent PCR protocol, developed in this study, allowed to directly detect and enumerate S. thermophilus in human feces, excluding culture-based techniques or time consuming DNA isolation and purification procedures. Intestinal persistence of S. thermophilus was studied in feces of 10 healthy subjects given VSL-3 or yogurt. Streptococcal population was detected after 3 days of administration and persisted for 6 days after the treatment suspension. In the same trial, the colonization kinetics of B. infantis Y1 and B. breve Y8 were studied by amplification of colonies with the strain-specific primer sets InfY-BV.L/R and BreY-BV.R/L, showing a host-dependent transient colonization behaviour. PCR analysis of feces from 10 patients affected by inflammatory bowel diseases (IBD) and treated with VSL-3 for 2 months showed a colonization pattern of S. thermophilus, B. infantis Y1 and B. breve Y8 similar to that observed with the healthy subjects. [Copyright &y& Elsevier]

Abstract Aims: To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause ‘blown pack’ spoilage of vacuum-packed meat. Methods and Results: Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of ‘blown pack’ causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of ‘blown pack’ causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT . The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g-1 for non-enriched, and 102 CFU g-1 for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of ‘blown pack’ spoilage in two packs, and Cl. estertheticum as the causative agent in the third. Conclusions: The developed method can be used for rapid detection of ‘blown pack’ causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. Significance and Impact of the Study: The paper reports... [ABSTRACT FROM AUTHOR]

We sought to determine the SAG2 genotypes of Toxoplasma gondii associated with cases of acute human toxoplasmosis in England and Wales. The samples examined were collected from a wide range of cases including congenital infections, AIDS and immunosuppressed patients and were derived from a number of different tissues. Parasite DNA was detected by PCR amplification without the need for prior template purification, and SAG2 genotype was determined by both restriction enzyme analysis and direct DNA sequencing of the PCR amplification products. Parasites of both SAG2 type I and type II genotypes were seen with approximately equal frequency amongst the samples examined. Neither of these genotypes was found to be more frequently associated with a particular clinical presentation or sample tissue. Unexpectedly, we found clear evidence of mixed (SAG2 type I+type II) infections in approximately the same number of samples as were seen to be associated with either type I or II alone. Our use of direct DNA sequencing rather than simple restriction analysis was essential for the detection of mixed infections since incomplete restriction digestion of samples containing a single parasite type was occasionally observed. It is possible that the presence of more than one type of parasite in single samples might be related to our recent demonstration that mixtures of SAG2 type I and type II parasites are present in a significant proportion of commercial meat preparations. Moreover, the presence of mixed infections in single patients might offer a direct molecular method of assessing risk factors for infection. [Copyright &y& Elsevier]

Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil). In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked. Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample. Consequently, preparation of samples prior to PCR depended upon sample origin. Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful. DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R. solanacearum in environmental samples. However, the commercial DNA extraction QIAamp® kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R. solanacearum were inefficient. This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R. solanacearum populations in their natural habitats. [Copyright &y& Elsevier]