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Pyrosequencing is a real-time DNA sequencing technique generating short reads rapidly and inexpensively. This technology has the potential advantage of accuracy, ease-of-use, high flexibility and is now emerging as a popular platform for microbial typing. Here, we review the methodology and the use of this technique for viral typing, bacterial typing, and fungal typing. In addition, we describe how to use multiplexing for accurate and rapid typing.
Oxford Nanopore Technology (ONT) enables rapid, portable pathogen identification and antimicrobial resistance (AMR) detection, but the reliability of downstream genomic analyses is highly dependent on DNA extraction quality, particularly in resource-limited settings. This study comparatively evaluated four portable bacterial DNA extraction protocols derived from three commercial kits to determine their impact on nanopore sequencing performance, bioinformatics workflow completion, and field deployability. Six gram-negative bacterial isolates (Escherichia coli, n = 4; Pseudomonas sp., n = 1; and Salmonella sp., n = 1) were processed using four extraction protocols: SwiftX DNA, SwiftX DNA with proteinase K (ProtK), SwiftX ParaBact, and NucleoSpin Microbial. Twenty-four resulting DNA extracts were sequenced on a single multiplexed MinION R10.4.1 flow cell. Sequencing data were analyzed using validated Galaxy-based generic and species-specific pipelines. Workflow completion was defined as successful progression through quality control, assembly, virulence, plasmid, and AMR detection modules. DNA purity varied substantially by extraction protocol and was strongly associated with successful workflow completion (Kruskal-Wallis, P = 0.0006). Accordingly, NucleoSpin Microbial achieved 100% workflow completion, and SwiftX ParaBact achieved 83%, while both SwiftX DNA-based protocols failed to complete full workflows. Importantly, key AMR genes required to classify isolates as multidrug-resistant were consistently detected using both NucleoSpin Microbial and SwiftX ParaBact extractions. However, NucleoSpin Microbial assemblies showed significantly higher contiguity and enabled a broader, more complete detection of virulence factors, pathogenicity islands, plasmid replicons, and accessory AMR genes, reflecting enhanced genomic resolution.IMPORTANCERapid whole-genome sequencing is increasingly used to detect antimicrobial resistance and guide public health responses, but its reliability depends strongly on how bacterial DNA is extracted. In this study, we have shown that DNA extraction method choice has a major impact on Oxford Nanopore sequencing performance across clinically relevant gram-negative bacteria. While silica column-based extraction maximized genomic completeness and analytical depth, paramagnetic bead-based reverse purification offered superior portability with sufficient resolution for frontline AMR surveillance. These findings highlight a practical trade-off between field deployability and high-resolution genomic characterization in low-resource settings.
Fluoroquinolones are broad-spectrum antimicrobials highly effective in the treatment of a wide variety of clinical infections. Salmonella gastroenteritis is usually only treated with fluoroquinolones when the patient is elderly or immunocompromised. Fluoroquinolones are also used for the treatment of systemic Salmonella infection or for long-term salmonella carriage. Resistance to quinolones is commonly mediated by point mutations within the topoisomerase genes gyrA and parC. Pyrosequencing technology is a DNA sequencing method using 'sequencing by synthesis' and is suitable for the rapid detection of single nucleotide polymorphisms (SNPs). One hundred and ten Salmonella enterica isolates, representing 18 different serotypes, were used in this study. One hundred and four isolates had ciprofloxacin MICs of 0.25-32 microg/mL; the remaining six were ciprofloxacin-sensitive (ciprofloxacin MIC<or=0.125 microg/mL). PCR amplification of the quinolone resistance-determining regions of gyrA and parC was performed and the resulting amplicons subjected to a Pyrosequencing protocol using cyclic dispensation of nucleotides. Amino acid substitutions at S83 and D87 were detected in gyrA and at T57 and S80 in parC. Silent mutations were also identified at G81 of gyrA and at V67, H75 and H77 of parC. This is the first report of a Pyrosequencing assay being used to detect mutations in genes conferring quinolone resistance. The Pyrosequencing technology is a rapid and reliable alternative to current methods for identification of gyrA and parC mutations in S. enterica.
Helicase-dependent amplification (HDA) is an isothermal in vitro DNA amplification method based upon the coordinated actions of helicases to separate double-stranded DNA and DNA polymerases to synthesize DNA. Previously, a mesophilic form of HDA (mHDA) utilizing the Escherichia coli UvrD helicase, DNA polymerase I Klenow fragment, two accessory proteins, MutL and single-stranded DNA-binding protein (SSB), was developed (1). In an effort to improve the specificity and performance of HDA, we have cloned and purified a thermostable UvrD helicase (Tte-UvrD) and the mutL homolog (Tte-MutL) from Thermoanaerobacter tengcongensis. Characterization of the Tte-UvrD helicase shows that it is stable and active from 45 to 65 degrees C. We have found that the Tte-UvrD helicase unwinds blunt-ended DNA duplexes as well as substrates possessing 3'- or 5'-ssDNA tails. Tte-UvrD was used to develop athermophilichelicase-dependent amplification (tHDA) system to selectively amplify target sequences at 60-65 degrees C. The tHDA system is more efficient than mHDA, displaying heightened amplification sensitivity without the need for the MutL and SSB accessory proteins. Amplification independent of MutL corresponds with studies demonstrating that maximal Tte-UvrD helicase activity does not require the mutL homolog. The tHDA system allows for rapid amplification and detection of targets present in genomic DNA. The expeditious nature and simplistic design of the tHDA platform makes the technology ideal for use in diagnostic applications requiring rapid identification of organisms at the point-of-need.
OBJECTIVE: The present study assessed the ability of a rapid chemiluminescent DNA probe assay to detect bacterial growth in blood culture specimens before their detection by continuous-monitoring blood culture (CMBC) systems. STUDY DESIGN: Three newborn intensive care units, which are members of the National Institute of Child Health and Human Development Neonatal Research Network, participated in this study. Each center employs an automated CMBC system against which the DNA probe was compared. A total of 1700 blood cultures were analyzed. A 3-ml aliquot of culture medium was removed from each culture during early incubation, processed, and analyzed by the DNA probe. RESULTS: Of 1700 blood cultures, 130 (7.6%) were detected positive by a CMBC system. Coagulase-negative staphylococci (CNS) were present in 90 (69%) of the positive cultures, and 26 (29%) were detected by the DNA probe. Other organisms accounted for the remaining 40 positive cultures, and 31 (78%) of these were detected by the probe assay. Seventy-six percent of all positive cultures were detected by a CMBC system within 24 hours. Ninety-eight percent of all positive cultures were detected by a CMBC system within 48 hours. Of the two organisms that grew in a CMBC system beyond 48 hours, both were CNS and one of these was considered a contaminant. Therefore, 99% of clinically significant organisms were detected by a CMBC system within 48 hours. CONCLUSION: This DNA probe is not sufficiently sensitive to be clinically useful; however, automated CMBC systems are sufficiently sensitive to aid in clinical decision-making regarding the continuance or discontinuance of antibiotic therapy following 48 hours of culture incubation.
This paper presents an overview of how microsystem technology tools can be applied to the development of rapid, out-of-laboratory measurement capabilities for the determinations of toxigenic fungi and mycotoxins in foodstuffs. Most of the topics discussed are all under investigation within the European Commission-sponsored project Good-Food (FP6-IST). These are DNA arrays, electronic noses and electronic tongues for the detection of fungal contaminants in feed, and biosensors and chemical sensors based on microfabricated electrode systems, antibodies and novel synthetic receptors for the detection of specific mycotoxins. The approach to resolution of these difficult measurement problems in real matrices requires a multidisciplinary approach. The technology tools discussed can provide a route to the rapid, on-site generation of data that can aid the safe production of high-quality foodstuffs.
The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.
New possibilities to modify function and direct repair in the central nervous system (CNS) have been established by the merger of gene transfer technology with neural transplantation. Rapid advances in viral-mediated DNA-delivery procedures permit the study of novel gene expression in neurons and glial cells. Foreign genes, transferred by a virus vector, can be used to generate new cell lines, identify transplanted cells, and express growth factors or enzymes for neurotransmitter synthesis. In addition to CNS cell types, non-neural cells are also being studied with transgene technology in the nervous system. Functional effects have been obtained with grafts of genetically modified cells in animal models of several nervous system disorders, and the recent results set the stage for potential application of these techniques to human CNS gene therapy.
New technologies for multiple chemical synthesis of oligonucleotides and stepwise hybridization on a solid-phase support enable the rapid and cost-effective preparation of long duplex DNA regions. Will these new technologies usher in a new era in protein engineering?
Magnetizable solid phase technology was used to develop a method for the rapid purification of recombinant proteins expressed in Escherichia coli. We describe the purification of two recombinant DNA-binding proteins: the minimal DNA-binding domain of the oncoprotein Myb and full-length yeast TFIIIA. Both were purified in one step directly from an E. coli lysate by means of magnetizable phosphocellulose particles (PhosphoMagnaCel). All operations were performed in microcentrifuge tubes and could be completed within 15 min. High purity and excellent recovery of proteins active in sequence specific DNA-binding were obtained. The procedure allowed the simultaneous purification of eight mutant Myb-proteins within 30 min.
I have developed a novel rapid amplification of cDNA ends (RACE) technology that uses multistranded DNA formation mediated by the RecA protein. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary single-stranded DNA in the presence of RecA and exonuclease I. The possibility of applying this finding to the direct cloning of a 5'-RACE product onto a cDNA fragment, which does not require the use of restriction endonucleases, was explored. The results show that the terminal multistranded structure formed by the RecA-mediated reaction can be applied to RACE systems. Modifications to the RACE protocol to improve the effectiveness of the technique are also suggested.
Rapid advances in microfabrication, DNA and protein microarray and microfluidic technologies have enabled the development of fully-integrated, miniaturized systems. These so called 'laboratory-on-a-chip' (LOC) devices perform sample preparation (i.e. concentration, separation and purification) together with biochemical reactions and detection steps in a simple and automated manner. We believe LOC technology for environmental microbiology studies will have immediate impacts on microbial monitoring by achieving detection and identification within minutes at the single-cell level, and on microbial ecology by deepening the understanding of microbial community structure and diversity and correlating these with niche-specific functions within a micro space. In the long run, significant impacts are anticipated on environmental metagenomics and proteomics.
As the Human Genome Project is generating an avalanche of genetic information, molecular researchers and clinical practitioners are setting new criteria for evaluation of the links between newly discovered gene mutations and human disorders. These requirements necessitate the development of highly accurate and yet rapid automated systems for genetic testing. We describe the detection of a single nucleotide polymorphism (SNP) on the human Y chromosome with the fully automated, sensitive, and rapid system WAVE designed for DNA fragment analysis. This new technology, based on temperature-modulated liquid chromatography and a high-resolution matrix, offers new dimensions to the molecular biology research. The versatility of the WAVE makes the equipment a universal molecular biology stations for clinical and research facilities. The key aspects to setting the operating parameters are discussed.
The discovery of the structure of DNA was a necessary prerequisite for determining the sequence of DNA molecules. Technological advances have now made it possible to sequence DNA rapidly and has resulted in public databases with over 30 billion nucleotides of known sequence. The analysis of these data has lead to new fields of science and to amazing advances in our understanding of evolution.
BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed mainly in patients with high-risk or advanced hematologic malignancies and congenital or acquired aplastic anemias. In the context of the significant risk of graft failure after allo-HSCT from alternative donors and the risk of relapse in recipients transplanted for malignancy, the precise monitoring of posttransplant hematopoietic chimerism is of utmost interest. Useful molecular methods for chimerism quantification after allogeneic transplantation, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers. Such analyses can be performed regardless of donor's and recipient's sex. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent in situ hybridization (FISH) can be applied. METHODS: We compared different techniques for analysis of posttransplant chimerism, namely FISH and PCR-based molecular methods with automated detection of fluorescent products in an ALFExpress DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE). We used Spearman correlation test. RESULTS: We have found high correlation between results obtained from the PCR/ALF Express and PCR/ABI 310 Genetic Analyzer. Lower, but still positive correlations were found between results of FISH technique and results obtained using automated DNA sizing technology. CONCLUSIONS: All the methods applied enable a rapid and accurate detection of post-HSCT chimerism.
By application of PCR technology, Cytomegalovirus(CMV) DNA can be detected from clinical specimens, rapidly and with high sensitivity. PCR-based assays can detect CMV DNA from blood even prior to the onset of clinical symptoms, so qualitative PCR assays are used as an indicator for preemptive therapy in transplant recipients. But it is not always helpful in CMV infection because it cannot distinguish latent from active infection, meaning lower positive predictive value for clinical disease. In recent years, quantitative PCR assays are increasingly being utilized in the clinical setting to (i) identify individuals at risk of developing CMV disease(determination of clinical threshold), (ii) monitor viral response to antiviral treatment, (iii) predict individuals at risk of clinical relapse, and (iv) serve as an early indicator of antiviral resistance.
An initiative was launched in 1994 by the Special Programme for Research and Training in Tropical Diseases (TDR) of the WHO to analyse the genomes of the parasites Filaria, Schistosoma, Leishmania, Trypanosoma brucei and Trypanosoma cruzi. Five networks were established through wide publicity, holding meetings of key laboratories and developing proposals which were then reviewed by the Steering Committee of Strategic Research for financial support. The aim of the Programme was to use the platform of these networks to: (1) train scientists from tropical disease-endemic countries; (2) transfer technology and share material and expertise, thereby reducing costs and increasing efficiency; and (3) provide an information system that is accessible globally as soon as the results become available. The initial target was to produce a low-resolution genome map for each of the parasites, but it soon became evident that by using rapidly developing technologies, it might be feasible to complete DNA-sequence analysis for some of the parasites in the next decade, as discussed here by Alberto Carlos Frasch and colleagues, with particular focus on the T. cruzi genome initiative.
The typing of a radiation hybrid (RH) panel is generally achieved using a unique primer pair for each marker. We here describe a complementing approach utilizing IRS-PCR. Advantages of this technology include the use of a single universal primer to specify any locus, the rapid typing of RH lines by hybridization, and the conservative use of hybrid DNA. The technology allows the mapping of a clone without the requirement for STS generation. To test the technique, we have mapped 48 BAC clones derived from mouse chromosome 12 which we mostly identified using complex probes. As mammalian genomes are repeat-rich, the technology can easily be adapted to species other than mouse.