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Distribution
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
In order to distinguish between the viable and nonviable phage MS2, as well as norovirus and bacteria, a combined propidium monoazide-qPCR assay was proposed (Kim SY and Ko 2012). Differences in the enumeration of the phage MS2 with culture and RT-qPCR methods were analyzed by slow sand filtration at a pilot plant (Lodder et al. 2013). Propidium monoazide treatment coupled with long-amplicon qPCR assays were assessed for their ability to quantify infectious MS2 in pure cultures and following inactivation by a range of UV light exposures and chlorine doses (McLellan et al. 2016).
Human Noroviruses
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
G. Sanchez, W. Randazzo, D.H. D'Souza
To overcome this, different strategies have been tested, such as (1) pretreatment with nucleases or proteolytic enzymes prior to nucleic acid extraction, eliminating the signal of nucleic acids belonging to damaged or inactivated virions82,83; and (2) pretreatment with nucleic acid intercalating dyes such as ethidium monoazide (EMA) or propidium monoazide (PMA).84 Parshionikar et al.85 first tested HNoVs with this methodology based on the ability of the intercalating dye to penetrate only into compromised capsids, to covalently bind the RNA after exposure to strong visible light, and finally to inhibit the PCR amplification. Only recently, PMAxx combined with RT-qPCR has successfully been applied to discriminate between infectious and noninfectious HNoVs in vegetables and irrigation waters.28 Escudero-Abarca et al.86 also reported that a SYBRGreen PMA-RT-qPCR assay, but not a Taqman RT-qPCR, distinguished between infectious and thermally treated HNoV GI.
Alternaria
Published in Dongyou Liu, Laboratory Models for Foodborne Infections, 2017
Alicia Rodríguez, Andrea Patriarca, Mar Rodríguez, María Jesús Andrade, Juan José Córdoba
As an alternative to traditional methods, nucleic-acid-based techniques are being increasingly applied to examine the taxonomic relationships among Alternaria species. Most of them have been focused on small-spored catenulate Alternaria, which show little resolution in their molecular phylogeny. However, cladistics analyses of “housekeeping genes” commonly used for other genera, such as the mitochondrial large subunit (mtLSU) ribosomal DNA, internal transcribed spacer (ITS), β-tubulin, translation elongation factor α, calmodulin, actin, chitin synthetase, etc., failed to discriminate among the small-spored species, except for the A. infectoria species group.42–45 There are also genomic techniques to detect, identify, and quantify toxigenic moulds in foodstuffs. So far, there are no molecular methods based on genes involved in the Alternaria mycotoxin biosynthesis pathways; however, some methods, which have used unique conserved genes that distinguish toxigenic and nontoxigenic Alternaria spp., have been developed successfully. Several molecular methods have been developed to detect the presence of Alternaria spores and biomass in foods, such as the one reported by Zur et al.,46 a polymerase chain reaction (PCR)-based method with primers specific to the ITS1 and ITS2 of the 5.8S rRNA gene of Alternaria to detect its presence in commercial tomato products. The main inconvenience of these methods is that viable and nonviable cells cannot be distinguished, thus resulting in an overestimation of the amount of spores that can actually produce mycotoxin in a food product. More recently, Crespo-Sempere et al.47 developed a method including a pretreatment of samples with nucleic-acid-intercalating dyes (propidium monoazide, PMA) prior to quantitative PCR. PMA selectively penetrates cells with a damaged membrane, inhibiting DNA amplification during PCR. The method, based on a primer pair (Alt4 and Alt5) specific to Alternaria spp., allowed quantifying a detection limit of 102 spores/g on tomatoes. Even though the tomato matrix had a protective effect on the cells against PMA toxicity, reducing the efficiency to distinguish between viable and nonviable cells, the method is still a suitable tool for quantifying viable Alternaria cells, which could be useful for estimating potential risks of mycotoxin contamination. The main drawback of these nucleic-acid-based methods as well as of the traditional identification of Alternaria followed by evaluation of mycotoxin production is that they only give information about the negative potential effect derived of the Alternaria presence in foods. Although the latter techniques allow taking corrective actions to avoid presence of mycotoxin-producing Alternaria on foods, for a more appropriate investigation of foodborne Alternaria it is necessary to use laboratory animals or cell system models in which the effect of Alternaria extracts or Alternaria mycotoxin contaminated foods can be evaluated. This could allow estimating the real risk of the presence of Alternaria in foods.
Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics
Published in Gut Microbes, 2022
Xu Zhang, Krystal Walker, Janice Mayne, Leyuan Li, Zhibin Ning, Alain Stintzi, Daniel Figeys
Biobanked stools stored in the presence of preservation chemicals, such as glycerol, are commonly used for either microbiome assay, fermentation or FMT applications.13,17,18 Using the bacterial cell counting approach, Guerin-Danan et al. demonstrated that freezing in glycerol buffer had no effect on aerobes in fecal samples, while decreased the survival of anaerobes, but did not exceed the inter-individual variations.19 In a mouse FMT experiment, Ericsson et al. demonstrated that FMT using frozen feces performed similarly to fresh feces in maintaining the richness, diversity, and composition of donor microbiota.20 Utilizing a propidium monoazide (PMA)-based live bacteria-specific P18CR and sequencing approach, Papanicolas et al. showed that freeze-thaw reduced the microbial viability to ~20% in feces, although the composition of viable microbiota was not significantly altered.21 In addition, the exposure of fecal samples to ambient air further reduced viability and could lead to >10-fold reductions in the abundance of important commensal bacteria, such as Faecalibacterium prausnitzii.21 Therefore, biobanking of live microbiota needs to be systematically evaluated to determine whether the viability and functional activity of microbiota are maintained.
Strategic advancements and multimodal applications of biofilm therapy
Published in Expert Opinion on Biological Therapy, 2021
Molecular methods include polymerase chain reaction (q-PCR) used for access of total cells that indicate amplification causing quick process which allows the quantification of various species within single sample [29]. Quantification of active bacteria present in biofilm is carried out using qPCR is a substitute technique for bacterial culture growth [29]. This technique provides high access to active cells in presence of extracellular DNA and its derivatives formed from deceased cells. In order to prevent DNA quantification, the treatment of samples is performed using propidium monoazide (PMA) before DNA extraction. The molecule is administered inside the membrane-compromised cell further introduced between DNA bases causing interaction with eDNA. Furthermore, the visible light falls onto the sample causing stable covalent linkages between PMA and DNA. The process of DNA extraction leads to loss of altered DNA that prevents amplification during qPCR. PMA-qPCR interaction is applicable for counting active bacterial cells and quantifies each microbe in mixed-species biofilms. The demerits of molecular technique includes high cost, overemphasize the cell count in presence of eDNA and viability dependant on membrane cell integrity.
Profiling the microbial contamination in aviation fuel from an airport
Published in Biofouling, 2019
Dong Hu, Wenfang Lin, Jie Zeng, Peng Wu, Menglu Zhang, Lizheng Guo, Chengsong Ye, Kun Wan, Xin Yu
In recent years, based on the principles of PCR, high-throughput sequencing (HTS), which has revolutionized microbial ecology (Stefani et al. 2015), and quantitative PCR (qPCR) technology have been developed for accurate qualitative and quantitative determination of microbes in fuel environments (Galvao and Lutterbach 2014; Leuchtle et al. 2015; Martin-Sanchez, Gorbushina, Toepel 2016; de Azambuja et al. 2017; Radwan et al. 2018). However, it should be noted that the DNA-based PCR method can hardly distinguish viable and dead cells, which might exaggerate the real risks, especially for fuel with long-term stagnation in tanks that might contain lots of dead microbes. It is reported that propidium monoazide (PMA) can penetrate membrane-damaged cells, intercalate into DNA, and then prevent the amplification of DNA during PCRs, thereby distinguishing dead microbes from the viable ones (Slimani et al. 2012). However, due to the importance of aviation fuel which is related to the safety of aircraft, an accurate description of viable cells is more practical for assessing the microbial contamination in aviation fuel environments, and the PMA-PCR method may have good prospects for characterizing the contaminating microbes.