A multiplex magnetic capture hybridization and multiplex Real-Time PCR protocol for pathogen detection in seafood.

Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1-10(3)cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1 cfu/g, in enriched samples, and higher sensitivity (10(2)-10(3)cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.

Extraction of DNA from soil using nanoparticles by magnetic bioseparation.

AIMS: To develop a simple, rapid and inexpensive soil DNA extraction protocol. METHODS AND RESULTS: The protocol relies on the use of superparamagnetic silica-magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. CONCLUSIONS: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol reported here is simple and permits rapid isolation of PCR-ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.

Capture probe conjugated to paramagnetic nanoparticles for purification of Alexandrium species (Dinophyceae) DNA from environmental samples.

AIMS: To develop a rapid, cost-effective and selective Alexandrium DNA extraction procedure from environmental samples in order to provide good-quality template for the downstream PCR-based detection assay. METHODS AND RESULTS: In this study, we tested a DNA extraction method based on silica-coated, superparamagnetic nanoparticles conjugated to a DNA-capture sequence (probe) complementary to a specific region of 5.8S rDNA of the genus Alexandrium. Cultured Alexandrium catenella cells were used as the harmful algal bloom species for the DNA extraction. Then, a PCR assay was performed with primers specific for the genus Alexandrium to assess the specificity and sensitivity of the nucleic acid extraction method. This method was applied to both cultured and field samples, reaching in both cases a detection limit of one A. catenella cell. CONCLUSIONS: The results suggest that the use of probe-conjugated paramagnetic nanoparticles could be effective for the specific purification of microalgal DNA in cultured or environmental samples, ensuring sensitivity and specificity of the subsequent PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA extraction method optimized in this study represents a progress towards the rapid and efficient direct detection of Alexandrium cells in seawater monitoring. In fact, this method requires no other equipment than a magnet and a hybridization oven and, in principle, can be adapted to different toxic microalgal species and can be automated, allowing the processing of a high number of samples.

Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples.

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.

Complete characterisation of Tn5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism.

The complete genetic characterisation of Tn5530 in Burkholderia cepacia strain 2a (pIJB1) has been accomplished, indicating that it is a Tn3-like transposon with a complex structure bearing operons for the catabolism of 2,4-dichlorophenoxyacetate (2,4-D) and malonate. Tn5530 is terminated at both ends by the IS1071::IS1471 element and the 2,4-D- and malonate-dissimilatory operons are separated by a region encoding a putA and lrp gene and a gene encoding a chloride channel protein. The chloride channel protein may have a role in the expulsion of chloride ions liberated by the dissimilation of 2,4-D. In addition, a putative transposase with a high level of sequence similarity to those of plasmid pGH1 from Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. glycinea, and a transcription factor similar to those of the TetR family with low but significant levels of sequence similarity to those identified in a number of other organisms was observed. The entire Tn5530 sequence length, including the IS1071::IS1471 elements, was found to be 40,956bp, and pIJB1 was replicon-typed and otherwise characterised as being of the IncP-1beta subgroup, bearing merA and merD genes conferring resistance to mercuric chloride. The rate of uptake of 2,4-D by B. cepacia strain 2a was observed to proceed more readily at acid pH, suggesting involvement of the undissociated form of the compound. Uptake did not show saturation kinetics, was concentration-dependent, and appeared to occur in two stages; an initial accumulation followed by a linear second phase. Uptake could be inhibited by sodium azide but not by arsenate, N,N(')-dicyclohexylcarbodi-imide (DCCD) or carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting that it is not energy-dependent.

2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134.

2,4-Dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2,4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an Mr of approximately 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha-KG and dioxygen, with the production of one mol each of succinate, 2,4-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degrees C, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+, Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha-KG were 109 and 8.9 microM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha-KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents.

Application of magnetite and silica-magnetite composites to the isolation of genomic DNA.

Magnetite and silica-magnetite composites were used as adsorbents for the isolation of genomic DNA from maize kernels. Two methods are described for the preparation of silica-magnetite composites, both of which afford higher yields of genomic DNA than when using magnetite alone, or a commerically available kit. DNA isolated using silica-magnetite was suitable for use in further applications such as polymerase chain reaction amplification and enzyme digestion.

The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar.

The complete sequence of the Atlantic salmon (Salmo salar) mitochondrial genome has been determined. The entire sequence is 16665 base pairs (bp) in length, with a gene content (13 protein-coding, two ribosomal RNA [rRNA] and 22 transfer RNA [tRNA] genes) and order conforming to that observed in most other vertebrates. Base composition and codon usage have been detailed. Nucleotide and derived amino acid sequences of the 13 protein-coding genes from Atlantic salmon have been compared with their counterparts in rainbow trout. A putative structure for the origin of L-strand replication (O(L)) is proposed, and sequence features of the control region (D-loop) are described.

Comparison of three RNA amplification methods as sources of DNA for sequencing.

DNA products generated from a region of the measles virus genome by three RNA reverse transcription and amplification methods were cloned and sequenced, and the results were compared in order to evaluate the methods' relative fidelities. The methods were: (i) reverse transcription followed by a nested polymerase chain reaction (RT-nPCR), (ii) a combined RT-PCR using rTth polymerase and (iii) nucleic acid sequence-based amplification (NASBA). NASBA was followed by RT-PCR with rTth polymerase or RT using AMV reverse transcriptase to generate DNA products for cloning. Products from all three sets of reactions were cloned into a vector, pT7Blue, and 790 bp of cloned DNA were sequenced and analyzed for base changes to determine the error rates for each amplification method. Sequence analysis of cloned RT-nPCR products showed no errors, whereas cloned rTth mediated RT-PCR products possessed an error rate of 0.38% and cloned NASBA products 0.38%. Products generated by NASBA followed by RT-PCR with rTth polymerase possessed an error rate of 1.9%. The results indicated that cloned DNA products generated by RT-nPCRs possessed least errors and that for NASBA, RT of reaction products before cloning and sequencing was preferable to using RT-PCR.

Recent developments of magnetic beads for use in nucleic acid purification.

The performance of Magarose, an agarose-based bead containing a paramagnetic component has been evaluated. The anion exchanger DEAE-Magarose is effective at binding DNA from a crude cell lysate. The plasmid pBluescript was isolated from 1.5 ml Escherichia coli JM109 cell culture, following alkaline lysis yielding 8.2 micrograms high-quality DNA. Under similar binding conditions 21 micrograms of salmon sperm DNA bound to the ion exchangers. The affinity medium oligo-dT Magarose was demonstrated to bind 75 mumol of an oligo-dA probe/g of medium by hybridization. Under similar conditions mRNA could be isolated from a preparation of baby hamster cell total RNA. The magnetic susceptibility of Magarose is very high, facilitating the use of this separation technique for rapid batch chromatographic processes.

Measles virus RNA is not detected in inflammatory bowel disease using hybrid capture and reverse transcription followed by the polymerase chain reaction.

Recent epidemiological and immunohistochemical studies have indicated a possible link between measles virus and inflammatory bowel disease (IBD). The aim of this study was to use a sensitive and robust method for the detection of measles virus RNA in IBD and control clinical samples. Peripheral blood mononuclear cells and intestinal resection tissue from IBD and control patients were studied. Two methods were used to determine the presence of measles virus RNA: hybrid capture, using measles virus-specific oligonucleotides linked to paramagnetic solid-phase supports, was carried out on total cellular RNA to enrich for measles virus RNA sequences. Reverse transcription followed by the polymerase chain reaction (RT-PCR) using rTth DNA polymerase was employed for amplification of measles virus N-gene sequences amongst the enriched species. Total RNA was also used for RT-PCR of a housekeeping mRNA species to assess RNA quality. RT-PCR for another region of the measles genome (the haemagglutinin (H) gene) was also undertaken in order to confirm the results obtained using N-gene primers for analysis of these samples. None of the samples were positive for measles N- or H-gene RNA using RT-PCR. Positive control samples confirmed the sensitivity of the methods employed. These results show that either measles virus RNA was not present in the samples, or was present below the sensitivity limits known to have been achieved.

A novel plasmid pIJB1 possessing a putative 2,4-dichlorophenoxyacetate degradative transposon Tn5530 in Burkholderia cepacia strain 2a.

A 102-kb plasmid, pIJB1, was isolated from Burkholderia cepacia strain 2a, which is able to use 2,4-dichlorophenoxyacetate (2,4-D) as a sole carbon source, and a physical map of the plasmid has been established. It was observed that spontaneous loss of a 37-kb fragment of the plasmid after growth in nonselective medium occurred, generating a plasmid of diminished size, pIJB2. The deletion event is concomitant with the loss of the 2,4-D dissimilatory phenotype, indicating that at least some of the 2,4-D degradative genes are on the missing fragment. The missing fragment is flanked by two identical 4.3-kb insertion sequences (IS) and shows a typical composite transposon structure of 41-kb in size, designated Tn5530. The mutant plasmid pIJB2 possesses a single copy of the IS element.

Stereolithographic (SL) biomodelling in craniofacial surgery.

BACKGROUND: Stereolithographic (SL) biomodelling allows 3D CT to be used to generate solid plastic replicas of anatomical structures (biomodels). Case reports in the literature suggest that such biomodels may have a use in craniofacial surgery but no large series or assessment of utility has been reported. A prospective trial to assess the utility of biomodelling in craniofacial surgery has been performed. METHODS: Forty patients with complex craniofacial abnormalities were selected and 3D CT scanning performed. The data of interest was used to guide a laser to selectively polymerise photosensitive resin to manufacture SL biomodels. The biomodels were used for patient education, diagnosis and operative planning. An assessment protocol was designed to test the hypothesis that biomodels in addition to standard imaging had greater utility in the surgery performed than the standard imaging alone. RESULTS: Anecdotally surgeons found biomodelling useful in 40 complex craniofacial operations. The formal assessment of the first 10 cases suggested biomodels improved operative planning (image 76%, image with biomodel 97%, P < 0.01) and diagnosis (image 82.5%, image with biomodel 99.25%, P < 0.01). Surgeons estimated that the use of biomodels had reduced operating time by a mean of 16% and were cost effective at a mean price of $1100 AUS. CONCLUSION: Biomodelling was reported as an intuitive, user-friendly technology that facilitated diagnosis, operative planning and communication between colleagues and patients. Limitations of the technology were manufacturing time and cost.

New approaches to the isolation of DNA by ion-exchange chromatography.

The performance of different anion-exchange media have been compared for the isolation of plasmid DNA and genomic DNA from bacterial cells and human whole blood. Whatman DEAE-Magarose, based on an agarose bead containing a paramagnetic component, has been compared with prepacked gravity-flow columns containing a derivatised silica matrix. In each case the DNA isolation at various scales of operation was similar both in terms of yield and quality. The magnetic susceptibility of DEAE-Magarose is very high, facilitating the use of this separation technique for rapid flexible batch chromatographic processes, a limitation of the prepacked column techniques.

Improved manufacture and application of an agarose magnetizable solid-phase support.

A simple, semiautomated, nonhazardous procedure for the production of a magnetizable solid-phase support (MSPS) has been developed based on the extrusion of molten agarose-iron oxide mixtures, which enables manufacture of a range of differently sized spherical agarose-iron oxide beads. This system has enabled scale-up of an original manufacture procedure and reproducible preparation of kg quantities of MSPS suitable for biomolecular purifications. An improved protocol for the isolation of plasmid DNA directly from cell lysates using this MSPS, derivatized with diethylaminoethyl (DEAE) groups, is reported. This involves a modified alkaline lysis, followed by adsorption to and elution from the support, yielding plasmid DNA of a purity comparable with, or better than, other methods of plasmid isolation. Using the same procedure, plasmid DNA can be isolated from bacterial cell culture volumes of 1.5 mL and 100 mL with equal efficiency and purity.

Rapid, sensitive, microbial detection by gene amplification using restriction endonuclease target sequences.

The use of primers synthesized to eight class II restriction endonuclease target sequences, from Haemophilus parainfluenzae, Escherichia coli, Staphylococcus aureus, Salmonella infantis, Rhodobacter sphaeroides, Klebsiella pneumoniae, Bacillus amyloliquefaciens and Proteus vulgaris for single and multiplex PCR identification of the organisms is discussed. Results indicate that the method is sensitive and specific enough to detect single cells and attogram amounts of target DNA. It has also been demonstrated that the primers can be used in whole cell PCR for identification and whole cell PCR product recovery could be enhanced by the addition of gelatin or DMSO to PCR reaction mixtures. Other results have indicated that the method can be used for the definite identification of specific individuals present in mixed cultures or suspensions of organisms. The applicability of the method for detection of a specific strain within a group of closely related organisms has also been investigated and for that sequence/organism the results suggest that the proposed method is indeed very specific and discriminative. It is suggested that as more information becomes available regarding such sequences and their distribution, this approach could form the basis of a widescale, rapid, simple and cheap identification and/or typing system for bacteria.

Identification and sequencing of a novel insertion sequence, IS1471, in Burkholderia cepacia strain 2a.

A novel insertion sequence (IS), IS1471, has been identified which is inserted into IS element IS1071 possessed by plasmid pIJB1 in Burkholderia cepacia strain 2a. Nucleotide sequencing has revealed that IS1471 is 1112 bp in length and is flanked by 22/21-bp imperfect inverted repeats with a 3-bp duplication of the target sequence IS1471 contains a single open reading frame encoding a putative polypeptide of 345 amino acids with molecular mass of 39406 Da. Searches of DNA and protein sequence databases did not result in the detection of any homologous IS elements, suggesting that IS1471 is novel and may not belong to any known IS groups.

Electrophoretic karyotype of the amylolytic yeast Lipomyces starkeyi and cloning, sequencing and chromosomal localization of its TRP1 gene.

The genome of the amylolytic yeast strain Lipomyces starkeyi NCYC 1436 was analysed using contour-clamped homogeneous electric field gel electrophoresis (CHEF). The banding pattern under a variety of running conditions indicating the presence of 11 different chromosome-sized DNA molecules. The sizes of these chromosome bands were determined by comparison with chromosomes from standard strains of Schizosaccharomyces pombe and Saccharomyces cerevisiae. The chromosomal bands were estimated to be within the range 0.7-2.8 Mb, with the genome (excluding mitochondrial DNA) estimated at 15 Mb. The molecular cloning of the TRP1 gene, isolated from a genomic library of this strain, is also reported: the gene was present on a 6.5-kb Sau3A DNA fragment, and complemented the trpC gene of E. coli. The DNA sequence was determined (EMBL accession No. Z68292) and compared to other tryptophan biosynthetic genes encoding N-(5'-phosphoribosyl) anthranilate isomerase (PRAI) activity. The gene was also used as a probe in hybridization studies, and by this means, its chromosomal location was identified.

Magnetizable solid-phase supports for purification of nucleic acids.

A simple, small scale non-hazardous procedure for the production of magnetizable solid-phase support (MSPS) beads has been developed based on the extrusion of agarose/iron oxide mixtures. The MSPS beads were derivatized using various amine ligands. Derivatized MSPS beads were used to adsorb nucleic acids from aqueous solution and to separate RNA/DNA mixtures.