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.     

ERIC-PCR-based strain-specific detection of phenol-degrading bacteria in activated sludge of wastewater treatment systems

Aims:  To evaluate the use of Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR)-derived probes and primers to specifically detect bacterial strains in an activated sludge microbial community.
Methods and Results:  ERIC-PCR was performed on two phenol-degrading bacterial strains, Arthrobacter nicotianae P1-7 and Klebsiella sp. P8-14. Their amplicons were DIG labelled for use as probes and then hybridized with ERIC-PCR fingerprints. The results showed the distinct band patterns for both bacterial strains. Strain-specific PCR primers were designed based on the sequences of ERIC-PCR bands. The DNA of each of these strains was successfully detected from its mixture with activated sludge DNA, either by using their respective ERIC-PCR-based probes for hybridization or by using species-specific primers for amplification, with higher sensitivity by latter method.
Conclusions:  Two phenol-degrading bacterial strains were identified from a mixture of activated sludge by using ERIC-PCR-based methods.
Significance and Impact of the Study:  The study demonstrated that the bacteria, which have important functions in complex wastewater treatment microbial communities, could be specifically detected by using ERIC-PCR fingerprint-based hybridization or amplification.     

Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans

By using pulsed-field gel electrophoresis, we have separated the entire chromosome bands and examined the electrophoretic karyotypes of 27 strains of Candida albicans. The electrophoretic karyotype varied widely among these strains. Their chromosomal DNAs were resolved into 7-12 bands ranging in size from 0.42 to 3.0 Mb. Most of the separated chromosomal bands were assigned by eight cloned C. albicans DNA probes. These results suggest that the haploid number of C. albicans chromosomes is eight. Each of the probes hybridized specifically to one or two bands of similar size in most strains. With the exception of the MGL1 probe, when two bands were detected by one probe, the size of one of them was very conserved whilst the other was of fairly variable size. The sizes of the chromosome bands assigned by the MGL1 probe were much more variable. As C. albicans is considered to be a diploid organism, it is inferred that the karyotype polymorphism between strains is mainly derived from wide size heterogeneity in one of the homologous chromosomes. Furthermore, we have confirmed species-specific and strain-specific variation in medically important Candida species (C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata). Electrophoretic karyotype analysis is thus useful for species assignation. The TUB2 probe, encoding C. albicans beta-tubulin, hybridized to the chromosomal DNA of all the Candida species examined, but four C. albicans probes exhibited cross-species hybridization with C. stellatoidea only. The karyotype of C. stellatoidea seems to be within the range of the intraspecies variation observed in C. albicans.     

High-throughput molecular identification of fish eggs using multiplex suspension bead arrays

The location and abundance of fish eggs provide information concerning the timing and location of spawning activities and can provide fishery-independent estimates of spawning biomass. However, the full value of egg and larval surveys is severely restricted because many species' eggs and larvae are morphologically similar, making species-level identification difficult. Recent efforts have shown that nearly all species of fish may be identified by mitochondrial DNA (mtDNA) sequences (e.g. via 'DNA barcoding'). By taking advantage of a DNA barcode database, we have developed oligonucleotide probes for 23 marine fish species that produce pelagic eggs commonly found in California waters. Probes were coupled to fluorescent microspheres to create a suspension bead array. Biotin-labelled primers were used to amplify the mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal rRNA genes from individual fish eggs. The amplicons were then hybridized to the bead array, and after the addition of a reporter fluorophore, samples were analysed by flow cytometry with Luminex 100 instrumentation. Probes specifically targeted eggs that are abundant and/or from morphologically indistinguishable species pairs. Results showed that the 33 different probes designed for this study accurately identified all samples when PCR was successful. Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput and lower cost than DNA sequencing. The increasing availability of DNA barcode data for numerous fish faunas worldwide suggests that bead arrays could be developed and widely used for fish egg, larval and tissue identifications.     

Digoxigenin-labelled peptide nucleic acid to detect lactobacilli PCR amplicons immobilized on membranes from denaturing gradient gel electrophoresis

AIMS: To develop a digoxigenin (DIG)-labeled peptide nucleic acid (PNA) probe for the detection of Lactobacillus-related genera amongst eubacterial amplicons obtained from vaginal samples using denaturing gradient gel electrophoresis (DGGE) blots. METHODS AND RESULTS: Part of the 16S rRNA gene sequence was used as a target for the PNA probe. After confirming probe specificity using chromosomal DNA from species and isolates that have been detected in the urogenital tract, it was successfully used to detect lactobacilli amplicons generated using eubacterial-specific 16S rRNA gene-targeted primers from vaginal tract samples immobilized on membranes from DGGE. CONCLUSIONS: The Lactobacillus-specific PNA probe could distinguish between DNA fragments from lactobacilli in a DGGE gel from other bacterial species, including those that migrated to a similar position. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the DIG-labelled PNA probe on blots of eubacterial PCR products from DGGE gels can be used to specifically detect lactobacilli in complex vaginal samples.     

Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays

DNA microarrays that are used as end-point detectors for PCR assays are typically composed of short (15-25 mer) oligonucleotide probes bound to glass. When designing these detectors, we have frequently encountered situations where a probe would not hybridize to its complementary, terminally labeled PCR amplicon. To determine if failures could be explained by general phenomenon such as secondary structure, we designed a microarray to detect eight regions of the Escherichia coli 16S rDNA gene. We then amplified eight amplicons of different lengths using a biotin conjugated, antisense primer. Amplicons were then hybridized to the microarray and detected using a combination of signal amplification and fluorescence. In most cases, probe sequences complementary to the 5' region of the amplified products (sense orientation) did not hybridize to their respective amplicon. We tested for positional bias and showed that a biotin conjugated sense primer mirrored the same probe failures. Nick translated products, however, hybridized to all probes. Because nick translation generates many labeled fragments of random length, we concluded that this method disrupted secondary structure that otherwise prevented the amplicons from hybridizing to their respective probes. We also show that nick translation does not compromise detector sensitivity even when used with long PCR amplicons (ca. 1.5 kbp). Despite the increased cost of the nick translation, we concluded that this labeling strategy will reduce the time needed to design new assays as well as avoid possible false negatives during field applications. Alternative labeling strategies are also discussed.     

Multi-probe Real-Time PCR Identification of Four Common Candida Species in Blood Culture Broth

We developed a single-tube real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting Candida albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Primers were designed to amplify 18S rRNA gene of the genus Candida, and DNA probes were designed to hybridize two areas of the amplicons. The amplification curves and specific melting peaks of the probes hybridized with PCR product were used for definite species identifications. The reaction specificity was 100 % when evaluating the assay using DNA samples from 21 isolates of fungal and bacterial species. The assay was further evaluated in 129 fungal blood culture broth samples which were culture positive for fungus. Of the 129 samples, 119 were positively identified as: C. albicans (39), C. tropicalis (30), C. parapsilosis (23), C. glabrata (20), Candida spp. (5), and two samples containing mixed C. glabrata/C. albicans and C. glabrata/C. tropicalis. The five Candida spp. were identified by sequencing analysis as C. krusei, C. dubliniensis, C. aquaetextoris, and two isolates of C. athensensis. Of the ten samples which showed negative PCR results, six were Cryptococcus neoformans, and the others were Trichosporon sp., Rhodotorula sp., Fusarium sp., and Penicillium marneffei. Our findings show that the assay was highly effective in identifying the four medically important Candida species. The results can be available within 3 h after positivity of a blood culture broth sample.     

DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract

A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-associated bacterial species of different genera. Glycogen was used in these protocols to improve the precipitation of small concentrations of DNA in ethanol without affecting the sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large differences are most like due to the difference in cell lysis efficiency, since a competitive PCR experiment did not indicate any preference for gram negative, low G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis was performed to investigate the effect of both DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A higher diversity in fecal samples was observed with the bead beating method than with the Triton-X100 method. Bands in the bead beating method-derived DGGE profiles corresponding to bands of cloned sequences of the Clostridium coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were absent or had low intensity in the Triton X-100 method-derived profiles. The applicability of the bead beating method was further investigated by analyzing biopsy samples from the human colon which contain approximately 10(6) cells.     

Consensus multiplex PCR–restriction fragment length polymorphism (RFLP) for rapid detection of plant mitochondrial DNA polymorphism

A fast, simple, and efficient approach, termed consensus multiplex PCR–RFLP, was developed and employed to detect mitochondrial (mt)DNA variation in three orchid species, Spiranthes hongkongensis, S. sinensis , and S. spiralis . Using multiplex PCR, three pairs of consensus mitchondrial primers were added simultaneously into each reaction tube to amplify three nonoverlapping introns located in the NADH dehydrogenase genes. Fragment length differences in the multiplex PCR amplicons were directly detectable between S. spiralis and the other two species. Further restriction analysis of the multiplex PCR amplicons revealed sufficient mtDNA polymorphism, suitable for phylogenetic studies at the interspecific level. This approach is well suited for large-scale population surveys of mitochondrial genome diversity in plants. Additionally, the maternal mode of inheritance of organelle genomes renders this approach valuable for rapid identification of the origin and specific parentage of hybrid or allopolyploid species.     

Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.     

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

A real‐time PCR assay for the differentiation of Candida species

Aims: We established a real‐time PCR assay for the detection and strain identification of Candida species and demonstrated the ability to differentiate between Candida albicans the most common species, and also Candida parapsilosis, Candida glabrata, Candida tropicalis and Candida dubliniensis by LightCycler PCR and melting curve analysis. Methods and Results: The DNA isolation from cultures and serum was established using the QIAmp Tissue Kit. The sensitivity of the assay was ≥ 2 genome equivalents/assay. It was possible to differentiate all investigated Candida species by melting curve analysis, and no cross‐reaction to human DNA or Aspergillus species could be observed. Conclusions: The established real‐time PCR assay is a useful tool for the rapid identification of Candida species and a base technology for more complex PCR assays. Significance and Impact of the Study: We carried out initial steps in validation of a PCR assay for the detection and differentiation of medically relevant Candida species. The PCR was improved by generating PCR standards, additional generation of melting curves for species identification and the possibility to investigate different specimens simultaneously.     

Species-specific PCR-DGGE markers to distinguish Pyrenophora species associated to cereal seeds

Pyrenophora species, toxigenic cereal pathogens, and causal agents of leaf and kernel diseases, bring about economic and food safety concerns. Traditionally, Pyrenophora taxa have been identified microscopically after a period of incubation on culture media. In this study, a simple nested PCR-denaturing gel electrophoresis (DGGE) method was developed to detect, differentiate and identify six Pyrenophora species in plant tissues. A primer, specific to Pyrenophora species and able to amplify a fragment of the ribosomal RNA (rRNA), following first round amplification with universal ITS primers, was designed by reviewing Pyrenophora ribosomal DNA sequences deposited in GenBank. The specificity of the primer was assessed by submitting its sequence to the GenBank Basic Local Alignment Search Tool (BLAST) algorithm, and was also tested with DNA extracted from several ascomycetous, basidiomycetous, and zygomycetous taxa. No PCR product was obtained from non-Pyrenophora species. PCR amplification of DNA extracted from pure cultures of the different Pyrenophora species generated amplicons of an approximate 350bp. DGGE effectively separated between all six Pyrenophora amplicons. Subsequently, amplicons of known Pyrenophora species were used as molecular markers when Pyrenophora infected wheat seed was analyzed by PCR-DGGE. The molecular-based approach described herein can be used to identify different Pyrenophora species directly from infected plant material.     

Molecular profiling of diatom assemblages in tropical lake sediments using taxon-specific PCR and Denaturing High-Performance Liquid Chromatography (PCR-DHPLC)

Here we present a protocol to genetically detect diatoms in sediments of the Kenyan tropical Lake Naivasha, based on taxon-specific PCR amplification of short fragments (approximately 100 bp) of the small subunit ribosomal (SSU) gene and subsequent separation of species-specific PCR products by PCR-based denaturing high-performance liquid chromatography (DHPLC). An evaluation of amplicons differing in primer specificity to diatoms and length of the fragments amplified demonstrated that the number of different diatom sequence types detected after cloning of the PCR products critically depended on the specificity of the primers to diatoms and the length of the amplified fragments whereby shorter fragments yielded more species of diatoms. The DHPLC was able to discriminate between very short amplicons based on the sequence difference, even if the fragments were of identical length and if the amplicons differed only in a small number of nucleotides. Generally, the method identified the dominant sequence types from mixed amplifications. A comparison with microscopic analysis of the sediment samples revealed that the sequence types identified in the molecular assessment corresponded well with the most dominant species. In summary, the PCR-based DHPLC protocol offers a fast, reliable and cost-efficient possibility to study DNA from sediments and other environmental samples with unknown organismic content, even for very short DNA fragments.     

Rapid detection and species identification of Mycobacterium spp. using real-time PCR and DNA-microarray

Infections with mycobacteria are an important issue in public health care. Here we present a "proof-of-principle" concept for the identification of 37 different Mycobacterium species using 5' exonuclease real-time PCR and DNA microarray based on the region upstream of the 65 kDa heat shock protein. With our two PCR probes, one complementary to all mycobacteria species, the other specific for the M. tbc-complex, 34 species were properly classified by real-time PCR. After reamplification and hybridization to a DNA microarray, all species showed a specific pattern. All 10 blindly tested positive cultures revealed a positive real-time PCR signal with the genus probe. After reamplification and hybridization, six samples could unambiguously be identified. One sample showed a mixture of presumably three species-specific patterns and sequencing the 16S rRNA confirmed the presence of a mixture. The hybridization results of three specimens could not be interpreted because the signal to background ratio was not sufficient. Two samples considered as negative controls (LAL Reagent Water (Cambrex) and DNA of Candida albicans) gave neither a genus nor a M. tbc-complex positive PCR signal. Based on these results we consider our method to be a promising tool for the rapid identification of different mycobacteria species, with the advantage of possible identification of mixed infections or contaminations.     

DNA probes specific for Aeromonas hydrophila (HG1)

Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.     

Identification of Candida albicans by polymerase chain reaction amplification of CaYST1 gene intron fragment

A single pair of primers, deduced from the intron nucleotide sequence of the Candida albicans CaYST1 gene, was used in PCR analysis performed with both genomic DNA and whole cells of clinical isolates of Candida species and other microorganisms. All the clinical C. albicans isolates generated the expected 310 bp amplicon; other Candida species as well as laboratory strains belonging to other fungal genera failed to amplify any DNA fragment, except for Candida pseudotropicalis (amplicon of 1200 bp), Kluyveromices marxianus (amplicon of 1250 bp) and Cryptococcus neoformans (several amplicons longer than 1200 bp). Unusual C. albicans isolates from Africa also yielded the expected 310 bp amplicon. These results indicate that genes containing intron sequences may be useful to design species-specific primers for identification of fungal strains by PCR. The sensitivity of the method was evaluated for C. albicans genomic DNA by using both various DNA concentrations (224 ng to 2.7 pg) and different cell amounts (10(7); to 5 cells). The results obtained may be useful in earlier detection of candidiasis.     

Molecular Identification of <Emphasis Type="Italic">Candida orthopsilosis</Emphasis> Isolated from Blood Culture

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.