Clinical applications of quantitative real-time PCR in virology
Publication date: Available online 7 July 2015 Source:Methods in Microbiology Author(s): Julia Engstrom-Melnyk , Pedro L. Rodriguez , Olivier Peraud , Raymond C. Hein Since the invention of the polymerase chain reaction (PCR) and discovery of Taq polymerase, PCR has become a staple in both research and clinical molecular laboratories. As clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has PCR performance. Through optimisation, the present-day uses of real-time PCR and quantitative real-time PCR are enumerable. The technique, combined...
Source: Methods in Microbiology - July 7, 2015 Category: Microbiology Source Type: research
Artificial nucleic acid probes and their applications in clinical microbiology
Publication date: Available online 10 June 2015 Source:Methods in Microbiology Author(s): Alon Singer , Yi-Wei Tang Nucleic acid probes are ubiquitous in molecular diagnostics as their inherent attributes lend themselves extremely well for those purposes. With the aim of developing ever more capable diagnostics, the emphasis has been on designing smarter assays or improving the sensors themselves, enabling the development of more accurate and more sensitive molecular diagnostics. Recently, however, a number of groups have begun designing and utilising artificial nucleic acid probes which have been specifically engine...
Source: Methods in Microbiology - June 10, 2015 Category: Microbiology Source Type: research
MALDI-TOF mass spectrometry in the clinical microbiology laboratory; beyond identification
Publication date: Available online 21 May 2015 Source:Methods in Microbiology Author(s): Sören Schubert , Markus Kostrzewa Within less than a decade, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has become a gold standard for microbial identification in clinical microbiology laboratories. Besides identification of microorganisms, the typing of single strains as well as antibiotic and antimycotic resistance testing has come into focus in order to speed up the diagnostic process. Similarly to microbial identification, the main drivers are the reduction of cost per test an...
Source: Methods in Microbiology - May 21, 2015 Category: Microbiology Source Type: research
Gene amplification and sequencing for bacterial identification
Publication date: Available online 19 May 2015 Source:Methods in Microbiology Author(s): Susanna K.P. Lau , Jade L.L. Teng , Chi-Chun Ho , Patrick C.Y. Woo The most important task of the clinical microbiology laboratory is to accurately identify the responsible pathogens to species level. Phenotypic methods for bacterial identification are associated with many problems, such as strains with unusual biochemical profiles and slowly growing bacterial species. In the past few decades, gene sequencing has evolved to be an objective method for bacterial identification. Among the various gene targets, the 16S rRNA gene is...
Source: Methods in Microbiology - May 19, 2015 Category: Microbiology Source Type: research
Accurate whole-genome sequencing-based epidemiological surveillance of mycobacterium tuberculosis
Publication date: Available online 19 May 2015 Source:Methods in Microbiology Author(s): Hannes Pouseele , Philip Supply Efficient molecular-guided epidemiological control of tuberculosis is especially important, because of the complex epidemiology, the insidious transmission and the specific biological features of its etiologic agent Mycobacterium tuberculosis. The most used typing system for epidemiological tracing of the pathogen is MIRU-VNTR typing, which has been internationally standardised under two formats (standard 24-locus based and 4 hypervariable locus based), optionally combined with spoligotyping. This ...
Source: Methods in Microbiology - May 19, 2015 Category: Microbiology Source Type: research
Solid and suspension microarrays for microbial diagnostics
Publication date: Available online 14 May 2015 Source:Methods in Microbiology Author(s): Steve Miller , Ulas Karaoz , Eoin Brodie , Sherry Dunbar Advancements in molecular technologies have provided new platforms that are being increasingly adopted for use in the clinical microbiology laboratory. Among these, microarray methods are particularly well suited for diagnostics as they allow multiplexing, or the ability to test for multiple targets simultaneously from the same specimen. Microarray technologies commonly used for the detection and identification of microbial targets include solid-state microarrays, electro...
Source: Methods in Microbiology - May 15, 2015 Category: Microbiology Source Type: research
Chapter 14 Continuing Importance of the “Phenotype” in the Genomic Era
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): Peter Kämpfer With the growing importance of whole-genome sequence data in the classification and identification of prokaryotes, i.e., the “genotype”, the importance of the phenotype seems to become less and less important. However, the phenotype, i.e., the entity of expressed information, is the driving force of evolution both in a given cellular context and in the environmental context. The information laid down in single genes, gene clusters and genomes may only be “visible” in a more or less “complex environment”, with the cel...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
Chapter 13 MALDI-TOF Mass Spectrometry Applied to Classification and Identification of Bacteria
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): Peter Schumann , Thomas Maier Since the introduction of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for classification and identification of prokaryotes approximately 20 years ago, this technique has found increasing application in medical diagnostics, pharmaceutical quality control and environmental research as well as in veterinary medicine and the food industry. MALDI-TOF MS has become a promising alternative to conventional identification techniques and is even beginning to outcompete phenot...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
Chapter 12 Bacterial Typing and Identification By Genomic Analysis of 16S–23S rRNA Intergenic Transcribed Spacer (ITS) Sequences
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): Volker Gürtler , Gangavarapu Subrahmanyam , Malathi Shekar , Biswajit Maiti , Indrani Karunasagar The rrn operon is the basic building block of all bacteria found in 1–15 copies per bacterial genome in all bacteria. All bacterial genomes contain rrn operons that contain four basic components: (i) 16S rRNA gene; (ii) 16S–23S intergenic transcribed spacer (ITS); (iii) 23S rRNA gene; (iv) 5S rRNA gene. The chapter “Bacterial Typing and Identification By Genomic Analysis of 16S–23S rRNA Intergenic Transcribed Spacer (ITS) Sequences”...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
Chapter 11 Multi-locus Sequence Analysis Taking Prokaryotic Systematics to the Next Level
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): Xiaoying Rong , Ying Huang Multi-locus sequence analysis (MLSA) is now established as a powerful phylogenetic approach for defining the taxonomic structure and evolutionary history of diverse bacteria, and for exploring population parameters that shape speciation. In this chapter, the emphasis is on practical procedures, notably MLSA schemes designed to clarify relationships within specific genera. MLSA uses data retrieved from fragmented nucleotide sequences of housekeeping genes (normally five to seven) spread across the chromosome, thereby...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
Chapter 10 Multi-Locus Sequence Typing and the Gene-by-Gene Approach to Bacterial Classification and Analysis of Population Variation
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): Alison J. Cody , Julia S. Bennett , Martin C.J. Maiden For nearly 30 years, 16S rRNA gene sequencing has been a fundamental tool for identification and cataloguing of bacterial diversity, but the diversity at this locus lacks the resolution to distinguish closely related bacteria. Multi-locus sequence typing (MLST) established the utility of a portable, gene-by-gene approach to population analyses, using both allelic and nucleotide sequence data that catalogue variation at seven housekeeping loci; however, it did not provide sufficient discr...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
Chapter 9 Reconciliation Approaches to Determining HGT, Duplications, and Losses in Gene Trees
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): Olga K. Kamneva , Naomi L. Ward Bacterial genome content varies greatly, even between closely related species, due to processes such as gene duplication, loss, and horizontal gene transfer (HGT). New genes derived from duplication or HGT give rise to new molecular functions within microbial genomes. Therefore, an in-depth understanding of gene family evolution is fundamental to bacterial genome annotation and gene function prediction. The genomic content of ancestral bacterial species is also of interest. This unit provides a general introduc...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
Chapter 8 Identification of Conserved Indels that are Useful for Classification and Evolutionary Studies
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): Radhey S. Gupta Phylogenetic treeing approaches for the classification of organisms have a number of important limitations. The most significant of these drawbacks is that different groups/taxa are distinguished from each other primarily on the basis of their branching in the trees and, in most cases, no unique property of any kind (viz. biochemical, molecular, structural or physiological) is known that is specifically shared by members of different taxa. Analyses of genome sequences are leading to discovery of multiple kinds of molecular mark...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
Chapter 7 Whole-Genome Sequencing for Rapid and Accurate Identification of Bacterial Transmission Pathways
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): Simon R. Harris , Chinyere K. Okoro The advent of DNA sequencing technologies with the capability to sequence the entire genomes of large numbers of bacteria in clinically relevant timescales has opened up the opportunity of using whole-genome sequencing as an epidemiological tool. With costs of sequencing also reducing, we are on the verge of a genomic epidemiology revolution that will provide a method that is both more widely applicable and more detailed than any other current bacterial typing technique. However, the wealth of information p...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
Chapter 6 Whole-Genome Analyses Average Nucleotide Identity
Publication date: 2014 Source:Methods in Microbiology, Volume 41 Author(s): David R. Arahal Average nucleotide identity (ANI) was proposed almost 10 years ago as a means to compare genetic relatedness among prokaryotic strains. It was found that values around 95% corresponded to the 70% DNA–DNA hybridization cut-off value that is widely used to delineate archaeal and bacterial species. ANI calculations are one of the many aspects and approaches that can be derived from comparative genomic data and used for taxonomic purposes. Here, an overview about the impact and current usage of ANI values is given together with d...
Source: Methods in Microbiology - December 4, 2014 Category: Microbiology Source Type: research
