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Molecular methodologies providing data on viral concentration and infectivity have been successfully used in environmental virology, supporting quantitative risk assessment studies. The present study aimed to assess human mastadenovirus (HAdV) intact particles using a derivative of propidium monoazide associated with qPCR (PMAxx-qPCR) in aquatic matrices. Initially, different concentrations of PMAxx were evaluated to establish an optimal protocol for treating different naturally contaminated matrices, using 10 min incubation in the dark at 200 rpm at room temperature and 15 min of photoactivation in the PMA-Lite™ LED photolysis device. There was no significant reduction in the quantification of infectious HAdV with increasing concentration of PMAxx used (20 µM, 50 µM, and 100 µM), except for sewage samples. In this matrix, a reduction of 5.01 log of genomic copies (GC)/L was observed from the concentration of 50 µM and revealed 100% HAdV particles with damaged capsids. On the other hand, the mean reduction of 0.51 log in stool samples using the same concentration mentioned above demonstrated 83% of damaged particles eliminated in the stool. Following, 50 µM PMAxx-qPCR protocol revealed a log reduction of 0.91, 0.67, and 1.05 in other samples of raw sewage, brackish, and seawater where HAdV concentration reached 1.47 × 104, 6.81 × 102, and 2.33 × 102 GC/L, respectively. Fifty micrometers of PMAxx protocol helped screen intact viruses from different matrices, including sea and brackish water.

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Introdução A hanseníase é uma doença infecciosa crônica, causada peloMycobacterium leprae, que se manifesta na pele e pode invadir o sistema nervoso periférico do paciente. O cultivo de seu agente etiológico em meios de cultura artificiaisou celulares ainda é um desafio e obstáculo para estudos relacionados à sua microbiologia. Para avaliar a viabilidade de células bacterianas, utiliza-se corantes fluorescente, como a monoazida de propídeo (PMA). O corante penetra somente nas células que estão com a membrana celular comprometida e reage com fração de hidrocarboneto a fim de resultar em uma modificação permanente do DNA. Objetivo Padronizar a utilização do corante monoazida de propídeo (PMAxx™) em combinação com a técnica de reação em cadeia da polimerase em tempo real (RT qPCR) para detecção da viabilidade do M. leprae. Metodologia Diferentes concentrações de PMAxx™ foram adicionadas a 250µl de suspensão bacilar purificada, proveniente de coxim plantar de camundongos previamente infectados. As amostras foram incubadas no escuro por diferentes tempos. Após a incubação, foram fotoativadas por exposição em lâmpada halógena de 650 W. Foram avaliados os parâmetros de concentração bacilar, tempo de incubação no escuro, tempo de exposição à luz e concentração do PMAxx™. O DNA do bacilo foi extraído utilizando-se um kit comercial e amplificadas por RT qPCR, com uso de primers específicos para as regiões Specific Repetitive Element (RLEP) do DNA de M. leprae Resultados Não houve diferença significativa no valor do ΔCt em nenhuma das concentrações de bacilos, indicando que não foi possível fazer a discriminação entre células vivas e inviáveis. O tempo ideal de incubação no escuro foi de 60 minutos, pois apresentaram uma diferenciação significativa do ΔCtvivo-morto com PMAxxTM e ΔCtmorto com e sem PMAxxTM. Em relação ao tempo de fotoativação, o maior valor de ΔCt observado foi submetido a sete minutos em exposição à luz. A concentração do PMAxxTM que apresentou uma diferenciação de ΔCt maior foi de 25µL. Discussão Os resultados mostram que o PMAxx™ tem uma boa eficácia com outras bactérias, mas ainda apresenta dificuldades em intercalar ao DNA de M. leprae. O uso do corante após análise com RT qPCR/RLEP para o bacilo é um método que ainda necessita de ajustes nos parâmetros como purificação da amostra, tempo de exposição e fotoativação. Esses dados ainda são preliminares e não inviabilizam a perspectiva de novos experimentos a partir dos ajustes nos parâmetros já avaliados.

Introduction Leprosy is a chronic infectious disease, caused by Mycobacterium leprae, which manifests itself in the skin and may invade the peripheral nervous system of the patient. Culturing its etiologic agent in artificial or cell culture media is still a challenge and obstacle for studies related to its microbiology. To assess the viability of bacterial cells, fluorescent dyes such as propidium monoazide (PMA) are used. The dye penetrates only cells with a compromised cell membrane and reacts with a hydrocarbon fraction to result in a permanent modification of the DNA Objective To standardize the use of the dye propidium monoazide (PMAxx™) in combination with the real-time polymerase chain reaction (RT qPCR) technique for detection of M. leprae viability Methodology Different concentrations of PMAxx™ were added to 250µl of purified bacillary suspension from plantar cushion of previously infected mice. The samples were incubated in the dark for different times. After incubation, they were photoactivated by exposure in a 650 W halogen lamp. The parameters of bacillary concentration, incubation time in the dark, light exposure time and concentration of PMAxx™ were evaluated. The bacillus DNA was extracted using a commercial kit and amplified by RT qPCR using specific primers for the Specific Repetitive Element (RLEP) regions of the M. leprae Results There was no significant difference in the ΔCt value at any of the bacilli concentrations, indicating that discrimination between live and non-viable cells was not possible. The optimal incubation time in the dark was 60 minutes, as they showed a significant differentiation of ΔClive-dead with PMAxxTM and ΔCtdead with and without PMAxxTM. Regarding photoactivation time, the highest value of ΔCt observed was subjected to seven minutes in light exposure. The concentration of PMAxxTM that showed a greater differentiation of ΔCt was 25µL Discussion The results show that PMAxx™ has good efficacy with other bacteria, but still presents difficulties in intercalating to M. leprae DNA. The use of the dye after analysis with RT qPCR/RLEP for the bacillus is a method that still needs adjustments in parameters such as sample purification, exposure time and photoactivation. These data are still preliminary and do not preclude the prospect of new experiments based on adjustments in the parameters already evaluated.

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Azospirillum brasilense is a plant growth promoting bacteria used as an inoculant in diverse crops. Accurate analytical methods are required to enumerate viable cells in inoculant formulations or in planta. We developed a quantitative polymerase chain reaction (qPCR) assay associated to propidium monoazide (PMA) to evaluate the cell viability of A. brasilense in inoculant and in maize roots. A. brasilense was grown in culture medium and was exposed to 50 â„ƒ. Maize roots were grown in vitro and harvested 7 days after inoculation. Quantification was performed by qPCR, PMA-qPCR, and plate counting. Standard curves efficiency values ranged from 85 to 99%. The limit of detection was 104 CFU per gram of fresh root. Enumeration obtained in maize roots by qPCR where higher than enumeration by PMA-qPCR and by plate counting. PMA-qPCR assay was efficient in quantifying inoculant viable cells and provides reliable results in a quickly and accurately way compared to culture-dependent methods.

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We report a specific and sensitive method to improve the coupling of propidium monoazide (PMA) with DNA derived from killed cells of Escherichia coli using UV light of 365 nm. UV light of three different intensities mainly 2.4 × 103, 4.8 × 103, and 7.2 × 103 µJ/cm2 was applied to E. coli cells each for 1, 3, and 5 min. PMA was found to be successfully cross-linked with the DNA from killed cells of E. coli at 4.8 × 103 µJ/cm2 in 3 min leading to the complete inhibition of PCR amplification of DNA derived from PMA-treated heat-killed cells. In spiked phosphate-buffered saline and potable water samples, the difference of the Cq values between PMA-treated viable cells and PMA-untreated viable cells ranged from -0.17 to 0.2, demonstrating that UV-induced PMA activation had a negligible effect on viable cells. In contrast, the difference of the Cq values between PMA-treated heat-killed cells and PMA-untreated heat-killed cells ranged from 8.9 to 9.99, indicating the ability of PMA to inhibit PCR amplification of DNA derived from killed cells to an equivalent as low as 100 CFU. In conclusion, this UV-coupled PMA-qPCR assay provided a rapid and sensitive methodology to selectively detect viable E. coli cells in spiked water samples within 4 h.

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Propidium monoazide (PMA) coupled with qPCR has been successfully used for specific quantification of viable bacteria cells in diverse matrices food. The present study aimed to develop PMA-qPCR assay for quantification of Lactobacillus paracasei viable cells in probiotic yoghurt. L. paracasei grown in culture medium was submitted to heat treatment at 60°C for different periods of time and probiotic yoghurt containing L. paracasei were prepared and stored at 4°C for 30days. The viable cells were quantified using qPCR and PMA-qPCR assays targeting tuf gene and also by plate counting. Standard curves were prepared and mean efficiency obtained was 94% and 96% (R2>0.98) to L. paracasei in culture medium and probiotic yoghurt stored one day, respectively. The limit of detection (LOD) for both samples was 104 genome copies, corresponding to 32.1pg of DNA. For viable cells quantification, standard curves Cq versus log CFU were plotted using mean CFU by plate counting of L. paracasei grown in culture medium and probiotic yoghurt. Results obtained for L. paracasei heat-treated cells were concordant by PMA-qPCR and plate count, CFU decreased as the heat treatment time increased, while qPCR count remained constant. L. paracasei enumerations obtained by qPCR for probiotic yoghurt stored one day and 30days were higher than enumerations by PMA-qPCR for the same samples. The plate count values were similar to CFU values obtained by PMA-qPCR. These results showed that PMA-qPCR is a powerful approach compared with culture-dependent methods for quantification of L. paracasei viable cells in yoghurt. PMA-qPCR allowed reliable obtained results much faster than plate counting.

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ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.(AU)

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ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

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The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

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The viability and resistance to simulated gastrointestinal (GI) conditions of Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 in synbiotic ice creams, in which milk was replaced by soy extract and/or whey protein isolate (WPI) with inulin, were investigated. The ice creams were showed to be satisfactory vehicles for La-5 and Bb-12 (populations around 7.5logCFU/g), even after the whole storage period (84days/-18°C). In all formulations, the propidium monoazide qPCR (PMA-qPCR) analysis demonstrated that probiotics could resist the in vitro GI assay, with significant survival levels, achieving survival rates exceeding 50%. Additionally, scanning electron microscopy images evidenced cells with morphological differences, suggesting physiological changes in response to the induced stress during the in vitro assay. Although all formulations provided resistance to the probiotic strains under GI stress, the variation found in probiotic survival suggests that GI tolerance is indeed affected by the choice of the food matrix.

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