Current Molecular Techniques for the detection of Microbial Pathogens

Abstract

Traditionally the detection of microbial pathogens in clinical, environmental or food samples has commonly needed the prelevation of cells by culture before the application of the detection strategy. This is done to increase cell number thereby overcoming problems associated with the sensitivity of classical detection strategies. However, culture-based methods have the disadvantages of taking longer, usually are more complex and require skilled personnel as well as not being able to detect viable but non cultivable microbial species. A number of molecular methods have been developed in the last 10 to 15 years to overcome these issues and to facilitate the rapid, accurate, sensitive and cost effective identification and enumeration of microorganisms which are designed to replace andyor support classical approaches to microbial detection. Amongst these new methods, ones based on the polymerase chain reaction and nucleic acid hybridization have been shown to be particularly suitable for this purpose. This review generally summarizes some of the current and emerging nucleic acid based molecular approaches for the detection, discrimination and quantification of microbes in environmental, food and clinical samples and includes reference to the recently developing areas of microfluidics and nanotechnology ''Lab-on-achip''.

PCR identification of toxic euglenid species Euglena sanguinea

“…At present, PCR-based methods are commonly used to detect and identify a variety of organisms, including toxic algae (Galluzzi et al 2007 ). The test presented herein also utilizes this methodology and allows for analysis of E .…”

Silicon, Silica and its Surface Patterning/Activation with Alkoxy- and Amino-Silanes for Nanomedical Applications

“…The surface functionalization of materials with biomolecules (e.g., nucleic acids, or antibodies or antibody [Ab] fragments) is of particular importance for many biomedical applications in vivo and in vitro; for example, immunoassays such as ELISA, the production of nucleic acid conjugated particles in the food industry, and in health, for the detection of pathogenic and food spoilage organisms and in smart contrast agents [97][98][99][100][101][102][103][104][105][106][107]. In the case of either flat surfaces, such as microarrays or particles, their surface activation ideally needs to be carried out in a controlled and reproducible fashion, so as to yield materials with optimized performance in the desired application and ones that will always work in the same way (see later).…”

Discriminating typical and atypical cystic fibrosis‐related bacteria by multiplex PNA‐FISH

“…Over the last 2 decades, there has been a significant progress on the development of rapid techniques (e.g., quantitative polymerase chain reaction-qPCR, 16S rRNA gene sequencing, denaturing/ temperature gradient gel electrophoresis-DGGE/TGGE, terminal restriction fragment length polymorphism-T-RFLP, and matrixassisted laser desorption ionization-time of flight mass spectrometry-MALDI-TOF MS) to detect, identify, and/or quantify microorganisms within polymicrobial communities (Bittar et al, 2008b;Fernandez-Olmos et al, 2012;Guss et al, 2011;Kirketerp-Moller et al, 2008;Lambiase et al, 2013;Malic et al, 2009;Rogers et al, 2003Rogers et al, , 2004Sibley et al, 2006). These promising molecular approaches have been developed to complement or even replace existing typical microbiological methods and other practices (Amann et al, 1995;Barenfanger et al, 1999;Call et al, 2003;Chadwick et al, 1998;Cleven et al, 2006;Hiyari and Bennett, 2011), highly improving the diagnosis of microbial species, providing more rapid, specific and sensitive systems than culture-dependent methods (Oosterheert et al, 2005), facilitating the identification of microorganisms present in biofilms (Bittar and Rolain, 2010;Galluzzi et al, 2007), and detecting abundant numbers of bacteria that do not grow under culture but are still viable (Hugenholtz, 2002;Oliver, 2005). It has been in this context that PNA FISH has emerged as a valuable tool for the specific and rapid detection of bacteria in polymicrobial communities without the need of cultivation (Almeida et al, 2010).…”

“…This time‐gap is extended for organisms that are fastidious, slow growing, noncultivable, or present as part of polymicrobial infections (Wolk and Dunne, ), as often occurs in CF. Additionally, these methods usually lead to the misidentification of unusual bacteria that improperly grow on unsuitable selective media and also fail in detecting microorganisms with compromised growth properties (e.g., after antibiotic therapy), and/or even anaerobic microorganisms, which require specific growth conditions (Bittar and Rolain, ; Bousbia et al, ; Galluzzi et al, ). Also, a range of different unusual species can grow in the same selective media used for the detection of other organisms, leading to misidentification (it is the case of I. limosus that grows in the Burkholderia cepacia selective medium) when using culturing methods (Bittar et al, ; Kidd et al, ; Spilker et al, ).…”