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Biological Contamination Control and Planetary Protection Measures as Applied to Sample Acquisition
Published in Yoseph Bar-Cohen, Kris Zacny, Advances in Extraterrestrial Drilling, 2020
James N. Benardini, Moogega Stricker, Kasthuri J. Venkateswaran
The advent of molecular technologies has dramatically increased the resolution and accuracy of detection of distinct microbial lineages in mixed microbial assemblages. Despite an expanding array of approaches for detecting microbes in a given sample, a rapid and robust means of assessing the differential viability of these cells, as a function of phylogenetic lineage, remains elusive. In several studies, pre-PCR propidium monoazide (PMA) treatment was coupled with downstream molecular (Illumina sequencing, pyrosequencing, and PhyloChip DNA microarray) analyses to better understand the frequency, diversity, and distribution of viable bacteria in spacecraft assembly cleanrooms. This also enables removal of dead bug body husks from the analysis. If dead bug body information is required, the difference between PMA-treated and non-PMA-treated samples will describe what percentage of microbial contamination was attributed to the dead bug bodies. Sample fractions not treated with PMA, which were indicative of the presence of both live and dead cells, yielded a great abundance of highly diverse microbial DNA sequences. In contrast, only 1%–10% of all of the sequencing reads, arising from a few robust microbial lineages, originated from sample fractions that had been pre-treated with PMA. The results of PhyloChip analyses of PMA-treated and -untreated sample fractions broadly agreed with those of DNA sequencing (Vaishampayan et al., 2013).
Nano-biosensors: A Custom-built Diagnosis
Published in Paula V. Messina, Luciano A. Benedini, Damián Placente, Tomorrow’s Healthcare by Nano-sized Approaches, 2020
Paula V. Messina, Luciano A. Benedini, Damián Placente
As we have emphasized in section 2.2.2 of chapter 2, the ‘‘Big Three’’ pathogens that span the spectrum of poverty related diseases (PRD) are: the human immunodeficiency virus (HIV), the Plasmodia spp. parasite which causes malaria, and the Mycobacterium tuberculosis (TB) bacterium. Together, they are leading the causes of mortality and morbidity in developing countries, accounting for more than half of all infant deaths. Over 95% of deaths linked to PRD are caused by a lack of proper diagnostics and treatments, due to insufficiencies in the health care infrastructure and cost constraints. Standard methods of pathogen detection, including cell culture, nucleic acid amplification, and enzyme-linked immunoassay are impractical because of the overburdened health care providers, the high volume of patients, work-flow and time limitations (Su et al. 2015). Thus, novel and easy to use pathogen detection platforms are emerging as powerful tools that meet the ASSURED criteria. Among them, the most prominent examples are included within microfluidic-based technology (Su et al. 2015). Microfluidic lab-on-a-chip (LOC) devices offer short processing times, reduced sample consumption, complex sample processing and handling of fluids, added to a portability and simplicity assimilated into a miniaturized arrangement (Foudeh et al. 2012). Several literature revisions of recently developed devices along with their respective advantages and limitations are available (Wang et al. 2013, Tay et al. 2016, Nasseri et al. 2018). At this point, we will examine the ways in which the tools at the sub-millimetre scale (microfluidics and nanotechnologies) can be addressed to meet the critical challenges to global public health. Fluorescence microscopy has, for a long time, been the standard method for TB detection in sputum samples, but it has been difficult to implement in the field. An integrated microfluidic system based on a fluorescence immune adsorption reaction for the capture, enrichment, and rapid detection of airborne Mycobacterium tuberculosis was proposed by W. Jing and co-workers (Jing et al. 2014). The whole detection consumed less than 50 min comprising 20 min of enrichment and 30 min of immunoreaction analysis. Likewise, Ka-U Ip et al. (Ip et al. 2018) proposed an integrated microfluidic device, including bacteria isolation, on-chip PCR and fluorescence detection, where the full analysis can be completed within 2 hours. The offered system can perform the entire detection of live bacteria from TB samples by the combination of a propidium monoazide (PMA) and real-time polymerase chain reaction (RT-PCR) with a limit of recognition of about 14 colony formation units CFU/reaction.
Formation mechanisms of viable but nonculturable bacteria through induction by light-based disinfection and their antibiotic resistance gene transfer risk: A review
Published in Critical Reviews in Environmental Science and Technology, 2021
Yiwei Cai, Jianying Liu, Guiying Li, Po Keung Wong, Taicheng An
In addition to LIVE/DEAD Baclight assay, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and real-time (quantitative) polymerase chain reaction (qPCR) are also common and useful methods for detecting VBNC bacteria (Foddai & Grant, 2020; Truchado et al., 2020; Wulsten et al., 2020). The principle of RT-qPCR-based detection is that the mRNA of dead cells will not be detectable after a short period, but the mRNA of live cells can still be assayed, so this method can be used to detect the presence of all viable cells (Dong et al., 2020; Yoon & Lee, 2020). The principle of qPCR-based detection to expose cells to specific compounds that penetrate membrane-permeable cells, causing irreversible damage to nucleic acids under subsequent light exposure, thereby strongly inhibiting PCR amplification of non-viable cells (Li et al., 2014). The end result is that only the DNA of cells with intact membranes will be amplified (Emerson et al., 2017). Zhou et al. applied propidium monoazide for cross priming amplification analysis to quickly detect foodborne VBNC E. coli O157:H7 (Zhou et al., 2020). Another study report a combined detection method for VBNC V. parahaemolyticus by using immunomagnetic separation with an improved propidium monoazide (Zhao et al., 2020). Truchado et al. combined ethidium monoazide with an improved propidium monoazide as DNA amplification inhibitors, followed by qPCR to detect VBNC cells in the wash water from fresh produce processing (Truchado et al., 2020). Baro et al. also used an optimized mixture of PMA and EMA combined with qPCR to detect VBNC Xylella fastidiosa induced by BP100 peptide conjugates (Baro et al., 2020). Although the above methods are sensitive, specific and rapid, it is necessary to combine the heterotrophic plate count method to know the number of VBNC bacteria.
Microbiology in Water-Miscible Metalworking Fluids
Published in Tribology Transactions, 2020
Frederick J. Passman, Peter Küenzi
Propidium monoazide-PCR (PMA-PCR or viability PCR) is a newer technique (196). PMA-PCR is in fact an effective tool to discriminate between intact and physically damaged cells in microorganisms. Therefore, analyzing PMA-treated DNA from populations reflects the structure of metabolically active/viable cells to a great extent.
Assays and enumeration of bioaerosols-traditional approaches to modern practices
Published in Aerosol Science and Technology, 2020
Maria D. King, Ronald E. Lacey, Hyoungmook Pak, Andrew Fearing, Gabriela Ramos, Tatiana Baig, Brooke Smith, Alexandra Koustova
VBNC and nonviable and nonculturable bioaerosols are classified through different methods than used for culturable microorganisms. These techniques include traditional microbiology methods, microscopy, immunoassays, molecular techniques and mass spectrometry (Table 2). Microscopy provides visual images and limited biological information of the collected particles that have to be collected undamaged. Immunoassays based on the analysis of specific or bulk proteins provide quantification of the biological composition (Womiloju et al. 2003). Nucleic acid based molecular techniques have been used for the identification of organisms at the species level (Hua and Tong 1992). Williams, Ward, and McCartney (2001) have applied conventional polymerase chain reaction (PCR) to analyze air samples for the presence of airborne mycobacteria and fungi commonly associated with adverse health effects. A PCR assay allows for the detection and identification of non-culturable airborne microorganisms (Peccia and Hernandez 2006) but does not allow for distinguishing between non-viable and viable microorganisms. To cover the gap between traditional microbial and molecular techniques for bioaerosol monitoring the culture-based analysis was combined with molecular analysis to increase the observed bacterial diversity (Hubad and Lapanje 2013). Currently, viability real-time PCR (RT-PCR) analysis is capable of accurate measurements of total microorganism concentrations in environmental samples with the ability to discriminate between live and dead cells by using propidium monoazide (PMA) (Nocker and Camper 2009). However, the toxicity of PMA at higher concentrations presents a limitation of the method (Taylor, Bentham, and Ross 2014). The advantage of RT-PCR is the ability of rapid sample quantification and species-specific identification (An, Mainelis, and White 2006). Microarrays, based on multiplexed 16S PCR reactions can identify a large number of genes if their sequence is known (Brodie et al. 2006). Whole genome sequencing and metagenomic analysis offer broad information about a bioaerosol sample (Boissy et al. 2014). The molecular assays offer the potential of real-time identification of single bioaerosol particles, however, the complexity of assays makes it difficult to detect single particles.