Electronic microarray analysis of 16S rDNA amplicons for bacterial detection

Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.     

Evaluation of the 16S and 12S rRNA genes as universal markers for the identification of commercial fish species in South Africa

The development of DNA-based methods for the identification of fish species is important for fisheries research and control, as well as for the detection of unintentional or fraudulent species substitutions in the marketplace. The aim of this study was to generate a comprehensive reference database of DNA sequences from the mitochondrial 16S and 12S ribosomal RNA (rRNA) genes for 53 commercial fish species in South Africa and to evaluate the applicability of these genetic markers for the identification of fish at the species level. The DNA extracted from all target species was readily amplified using universal primers targeting both rRNA gene regions. Sequences from the 16S and 12S rRNA genes were submitted to GenBank for the first time for 34% and 53% of the fish species, respectively. Cumulative analysis of the 16S rRNA gene sequences revealed mean conspecific, congeneric and confamilial Kimura two parameter (K2P) distances of 0.03%, 0.70% and 5.10% and the corresponding values at the 12S level were 0.03%, 1.00% and 5.57%. K2P neighbour-joining trees based on both sequence datasets generally clustered species in accordance with their taxonomic classifications. The nucleotide variation in both the 16S and 12S sequences was suitable for identifying the large majority of the examined fish specimens to at least the level of genus, but was found to be less useful for the explicit differentiation of certain congeneric fish species. It is recommended that one or more faster-evolving DNA regions be analysed to confirm the identities of closely-related fish species in South Africa.     

Identifying Fishes through DNA Barcodes and Microarrays

Background

International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.

Methodology/Principal Findings

This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.

Conclusions/Significance

Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.     

Applications of DNA barcoding to fish landings: authentication and diversity assessment

DNA barcoding methodologies are being increasingly applied not only for scientific purposes but also for diverse real-life uses. Fisheries assessment is a potential niche for DNA barcoding, which serves for species authentication and may also be used for estimating within-population genetic diversity of exploited fish. Analysis of single-sequence barcodes has been proposed as a shortcut for measuring diversity in addition to the original purpose of species identification. Here we explore the relative utility of different mitochondrial sequences (12S rDNA, COI, cyt b, and D-Loop) for application as barcodes in fisheries sciences, using as case studies two marine and two freshwater catches of contrasting diversity levels. Ambiguous catch identification from COI and cyt b was observed. In some cases this could be attributed to duplicated names in databases, but in others it could be due to mitochondrial introgression between closely related species that may obscure species assignation from mtDNA. This last problem could be solved using a combination of mitochondrial and nuclear genes. We suggest to simultaneously analyze one conserved and one more polymorphic gene to identify species and assess diversity in fish catches.     

Fluorescent oligonucleotide rDNA probes for specific detection of methane oxidising bacteria

Oligonucleotide probes targeting the 16S rRNA of distinct phylogenetic groups of methanotrophs were designed for the in situ detection of these organisms. A probe, MG-64, detected specifically type I methanotrophs, while probes MA-221 and MA-621, detected type II methanotrophs in whole cell hybridisations. A probe Mc1029 was also designed which targeted only organisms from the Methylococcus genus after whole cell hybridisations. All probes were labelled with the fluorochrome Cy3 and optimum conditions for hybridisation were determined. Non-specific target sites of the type I (MG-64) and type II (MA-621) probes to non-methanotrophic organisms are highlighted. The probes are however used in studying enrichment cultures and environments where selective pressure favours the growth of methanotrophs over other organisms. The application of these probes was demonstrated in the detection of type I methanotrophs with the MG-64 probe in an enrichment culture from an estuarine sample demonstrating methane oxidation. The detection of type I methanotrophs was confirmed by a 16S rDNA molecular analysis of the estuarine enrichment culture which demonstrated that the most abundant bacterial clone type in the 16S rDNA library was most closely related to Methylobacter sp. strain BB5.1, a type I methanotroph also isolated from an estuarine environment.     

Bacterial community associated with Pfiesteria-like dinoflagellate cultures

Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four ' Pfiesteria -like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the α-3 or SAR83 cluster.     

Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays

We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.     

Rapid identification of Aeromonas species using 16S rDNA targeted oligonucleotide primers: a molecular approach based on screening of environmental isolates

M. DORSCH, N.J. ASHBOLT, P.T. COX AND A.E. GOODMAN. 1994. Published 16S rDNA sequencing data for Aeromonas species were analysed and the validity of signature sequences derived from our investigations of these sequences was examined by sequencing the corresponding 16S rDNA regions of 67 environmental isolates from sewage effluents and receiving waters around Sydney and one clinical isolate, all previously classified as Aeromonas species. Species-specific probes for Aer. hydrophila and Aer. veronii were designed and tested in PCR assays and clearly discriminated these species from the other Aeromonas isolates as identified by 16S rDNA sequences.     

云南秋海棠属植物小志

描写了云南产秋海棠属6个新种1个新变种,它们是澜沧秋海棠、角果秋海棠、盈江秋海棠、粉叶秋海棠、蔓耗秋海棠、斜叶秋海棠、红毛香花秋海棠,补充描述了8个种及新命名1种,即四棱秋海棠、不显秋海棠、薄叶秋海棠、截裂秋海棠、长柔毛秋海棠、光叶秋海棠、变色秋海棠、假厚叶秋海棠、河口秋海棠。     

Karyotype analysis of four Vicia species using in situ hybridization with repetitive sequences

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S-25S and 5S rDNA, telomeres) and genus-specific satellite repeats (VicTR-A and VicTR-B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR-A and -B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied.     

蓝藻OscilatoriaceaerDNA16S-23S基因间隔区的序列分析及其分类学意义

采用末端终止法对蓝藻类颤藻科Ｏｓｃｉｌａｔｏｒｉａｓｐ．ｒＤＮＡ１６Ｓ－２３Ｓ基因间隔区进行了序列测定,获得了Ｏｓｃｉｌａｔｏｒｉａｓｐ．ｒＤＮＡ基因间隔区４２７个核苷酸,其中包含１个异亮氨酸ｔＲＮＡ基因（ｔＲＮＡＩｌｅ）。并通过计算机联网从国际分子生物学数据弹库中获取颤藻科其它种的ｒＤＮＡ基因间隔区序列,通过比较分析,从分子水平对颤藻科Ｏｓｃｉｌａｔｏｒｉａｃｅａｅ属间的某些分类学问题进行了讨论,并根据序列中核苷酸差异值探讨了颤藻科属间界定的分子标准。提出了ｒＤＮＡ基因间隔区是良好的分子标记,可用于“赤潮”或“水华”蓝藻专一性核酸分子探针的研制     

Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays

We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55°C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 × 10⁶ genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.     

Comprehensive aligned sequence construction for automated design of effective probes (CASCADE-P) using 16S rDNA

MOTIVATION: Prokaryotic organisms have been identified utilizing the sequence variation of the 16S rRNA gene. Variations steer the design of DNA probes for the detection of taxonomic groups or specific organisms. The long-term goal of our project is to create probe arrays capable of identifying 16S rDNA sequences in unknown samples. This necessitated the authentication, categorization and alignment of the >75 000 publicly available '16S' sequences. Preferably, the entire process should be computationally administrated so the aligned collection could periodically absorb 16S rDNA sequences from the public records. A complete multiple sequence alignment would provide a foundation for computational probe selection and facilitates microbial taxonomy and phylogeny. RESULTS: Here we report the alignment and similarity clustering of 62 662 16S rDNA sequences and an approach for designing effective probes for each cluster. A novel alignment compression algorithm, NAST (Nearest Alignment Space Termination), was designed to produce the uniform multiple sequence alignment referred to as the prokMSA. From the prokMSA, 9020 Operational Taxonomic Units (OTUs) were found based on transitive sequence similarities. An automated approach to probe design was straightforward using the prokMSA clustered into OTUs. As a test case, multiple probes were computationally picked for each of the 27 OTUs that were identified within the Staphylococcus Group. The probes were incorporated into a customized microarray and were able to correctly categorize Staphylococcus aureus and Bacillus anthracis into their correct OTUs. Although a successful probe picking strategy is outlined, the main focus of creating the prokMSA was to provide a comprehensive, categorized, updateable 16S rDNA collection useful as a foundation for any probe selection algorithm.     

Molecular organization of 5S rDNA in fishes of the genus Brycon.

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.     

Chromosomal mapping of 18S-28S and 5S rRNA genes by two-colour fluorescent in situ hybridization in six sturgeon species.

The number and distribution of the 18S-28S and 5S rRNA (rDNA) gene sequences were examined on mitotic chromosomes of six sturgeon species by two-colour in situ hybridization. Four of the six species, Huso huso, Acipenser stellatus, Acipenser sturio, and Acipenser ruthenus, with about 120 chromosomes, showed from six to eight 18S-28S rDNA signals, while 5S rDNA signals were on only one chromosome pair. The two species with 250-270 chromosomes, Acipenser baerii and Acipenser transmontanus, showed from 10 to 12 18S-28S sites and two chromosome pairs bearing 5S rDNA signals. In all examined species, the rather intense 5S rDNA signals apparently overlapped those of 18S-28S rDNA. These data support the diploid-tetraploid relationships between the two chromosome groups of sturgeons. The close association between the two rDNA families in species belonging to an ancestral fish order, such as Acipenseriformes, supports the hypothesis that the association represents a primitive condition.     

Sequence-based differentiation of strains in the Mycobacterium avium complex.

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.     

Conservation of the 5S-bearing chromosome pair and co-localization with major rDNA clusters in five species of Astyanax (Pisces,Characidae)

Major and 5S ribosomal genes have been localized in chromosomes from five fish species, genus Astyanax, using in situ hybridization (FISH) with 28S and 5S rDNA probes. In situ signals for the major rDNA co-localized with the 5S rDNA clusters in the pericentromeric region of one marker chromosome in all five species analyzed. The conserved localization of these two rDNA clusters in the five related Astyanax species was considered as indicative of a close relationship among them. The use of these molecular markers for elucidating evolutionary relationships among closely related taxa is discussed.     

Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.     

Differentiation of Lactobacillus helveticus,Lactobacillus delbrueckii subsp bulgaricus,subsp lactis and subsp delbrueckii using physiological and genetic tools and reclassification of some strains from the ATCC collection

Several physiological tests of glucose metabolism and genetic tools including species specific probes and 16S rDNA sequences were used to identify strains of L. helveticus and the group of L. delbrueckii with its three subspecies lactis, bulgaricus, and delbrueckii. These species are important for the milk industry as fermenting lactic acid bacteria. The identification procedure was applied to the different strains of these species available from the ATCC collection and allowed to reclassify part of them.     

Comparative sequence analysis and oligonucleotide probe design based on 23S rRNA genes of Alphaproteobacteria from North Sea bacterioplankton

Almost complete 23S rRNA gene sequences were obtained from 11 Alphaproteobacteria isolated from marine surface water of the German Bight. Five of the strains belong to the "marine alpha" group, a phylogenetic cluster which encompasses members of the genus Roseobacter and closely related bacteria. Phylogenetic sequence analysis based on 52 published as well as unpublished complete 23S rDNA sequences from Alphaproteobacteria including the newly obtained was in general consistent with the 16S rRNA gene sequence-derived phylogeny. 16S and 23S rRNA based phylogenies both showed a distinct cluster for strains associated with the "marine alpha" group. The suitability of both markers for the design of oligonucleotide probes targeting selected groups of Alphaproteobacteria was systematically evaluated and compared in silico. Six clusters of sequences covering different phylogenetic levels as well as two strains were selected in a case study. To compensate for the quantitative difference in the two data sets, the 16S rRNA dataset was truncated to sequences with an equivalent in the 23S rRNA data set. Our results show, that the overall number of phylogenetically redundant probes available could be more than doubled by extending probe design to the 23S rRNA. For small clusters of high sequence similarity and single strains, up to 8 times more discriminating binding sites were provided by the 23S rRNA.