10 results on '"viability-qPCR"'
Search Results
2. Verification and application of qPCR and viability-qPCR for Legionella monitoring in evaporative cooling systems complementing the conventional culture method.
- Author
-
Redwitz, J., Streich, P., Zamfir, M., Walser-Reichenbach, S.M., Seidel, M., Herr, C.E.W., Heinze, S., and Quartucci, C.
- Published
- 2024
- Full Text
- View/download PDF
3. Screening and identification of BP100 peptide conjugates active against Xylella fastidiosa using a viability-qPCR method
- Author
-
Aina Baró, Esther Badosa, Laura Montesinos, Lidia Feliu, Marta Planas, Emilio Montesinos, and Anna Bonaterra
- Subjects
Antimicrobial peptides ,Xylella fastidiosa ,Viability-qPCR ,PEMAX ,Plant pathogens ,Microbiology ,QR1-502 - Abstract
Abstract Background Xylella fastidiosa is one of the most harmful bacterial plant pathogens worldwide, causing a variety of diseases, with huge economic impact to agriculture and environment. Although it has been extensively studied, there are no therapeutic solutions to suppress disease development in infected plants. In this context, antimicrobial peptides represent promising alternatives to traditional compounds due to their activity against a wide range of plant pathogens, their low cytotoxicity, their mode of action that make resistance more difficult and their availability for being expressed in plants. Results Peptide conjugates derived from the lead peptide BP100 and fragments of cecropin, magainin or melittin were selected and tested against the plant pathogenic bacteria X. fastidiosa. In order to screen the activity of these antimicrobials, and due to the fastidious nature of the pathogen, a methodology consisting of a contact test coupled with the viability-quantitative PCR (v-qPCR) method was developed. The nucleic acid-binding dye PEMAX was used to selectively quantify viable cells by v-qPCR. In addition, the primer set XF16S-3 amplifying a 279 bp fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed by comparing v-qPCR viable cells estimation with conventional qPCR and plate counting. When cells were treated with peptide conjugates derived from BP100, the observed differences between methods suggested that, in addition to cell death due to the lytic effect of the peptides, there was an induction of the viable but non-culturable state in cells. Notably, a contact test coupled to v-qPCR allowed fast and accurate screening of antimicrobial peptides, and led to the identification of new peptide conjugates active against X. fastidiosa. Conclusions Antimicrobial peptides active against X. fastidiosa have been identified using an optimized methodology that quantifies viable cells without a cultivation stage, avoiding underestimation or false negative detection of the pathogen due to the viable but non-culturable state, and overestimation of the viable population observed using qPCR. These findings provide new alternative compounds for being tested in planta for the control of X. fastidiosa, and a methodology that enables the fast screening of a large amount of antimicrobials against this plant pathogenic bacterium.
- Published
- 2020
- Full Text
- View/download PDF
4. Development of a Long-Amplicon Propidium Monoazide-Quantitative PCR Assay for Detection of Viable Xanthomonas arboricola pv. pruni Cells in Peach Trees.
- Author
-
Panth M, Noh E, Schnabel G, and Wang H
- Subjects
- Real-Time Polymerase Chain Reaction methods, DNA, Bacterial genetics, Trees microbiology, Azides chemistry, Xanthomonas genetics, Xanthomonas isolation & purification, Propidium analogs & derivatives, Propidium chemistry, Plant Diseases microbiology, Prunus persica microbiology
- Abstract
Bacterial spot is one of the most serious diseases of peach caused by the pathogen Xanthomonas arboricola pv. pruni (XAP), leading to early defoliation and unmarketable fruit. The pathogen can overwinter in peach twigs and form spring cankers, which are considered the primary inoculum source for early season leaf and fruitlet infection. The amount of overwintering bacterial inoculum plays a critical role for the bacterial spot development, but no reliable quantification method is available. Thus, we developed a long-amplicon propidium monoazide (PMA)-quantitative PCR (qPCR) assay for specific detection of viable XAP cells. The optimized PMA-qPCR assay used 20 μM of PMAxx for pure bacterial suspensions and 100 μM for peach twig tissues. The Qiagen Plant Pro Kit with an additional lysozyme digestion step was the DNA extraction protocol that yielded the best detection sensitivity with the bacteria-spiked peach twig extracts. The PMA-qPCR assay was tested with different mixtures of viable and heat-killed XAP cells in pure bacterial suspensions and bacteria-spiked peach twig tissues. The results showed that this assay enabled sensitive, specific, and accurate quantification of viable XAP cells as low as 10
3 CFU/ml with the presence of up to 107 CFU/ml of dead XAP cells, while suppressing the amplification of DNA from dead cells. For mixtures of viable and dead cells, the PMA-qPCR results were linearly correlated with the predicted concentrations of viable XAP ( R2 > 0.98). Thus, the PMA-qPCR assay will be a suitable tool for quantifying overwintering XAP population on peach trees., Competing Interests: The author(s) declare no conflict of interest.- Published
- 2024
- Full Text
- View/download PDF
5. Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the Gardnerella-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis
- Author
-
Agnieszka Latka, Leen Van Simaey, Marijke Reynders, Piet Cools, Tess Rogier, Barbara Lebbe, Lorenzo Corsini, Christine Landlinger, and Mario Vaneechoutte
- Subjects
endolysin ,bacterial vaginosis ,biofilm ,Gardnerella ,viability-qPCR ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR.
- Published
- 2022
- Full Text
- View/download PDF
6. Screening and identification of BP100 peptide conjugates active against Xylella fastidiosa using a viability-qPCR method.
- Author
-
Baró, Aina, Badosa, Esther, Montesinos, Laura, Feliu, Lidia, Planas, Marta, Montesinos, Emilio, and Bonaterra, Anna
- Subjects
- *
PHYTOPATHOGENIC bacteria , *PHYTOPATHOGENIC microorganisms , *PEPTIDE antibiotics , *XYLELLA fastidiosa , *MELITTIN - Abstract
Background: Xylella fastidiosa is one of the most harmful bacterial plant pathogens worldwide, causing a variety of diseases, with huge economic impact to agriculture and environment. Although it has been extensively studied, there are no therapeutic solutions to suppress disease development in infected plants. In this context, antimicrobial peptides represent promising alternatives to traditional compounds due to their activity against a wide range of plant pathogens, their low cytotoxicity, their mode of action that make resistance more difficult and their availability for being expressed in plants. Results: Peptide conjugates derived from the lead peptide BP100 and fragments of cecropin, magainin or melittin were selected and tested against the plant pathogenic bacteria X. fastidiosa. In order to screen the activity of these antimicrobials, and due to the fastidious nature of the pathogen, a methodology consisting of a contact test coupled with the viability-quantitative PCR (v-qPCR) method was developed. The nucleic acid-binding dye PEMAX was used to selectively quantify viable cells by v-qPCR. In addition, the primer set XF16S-3 amplifying a 279 bp fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed by comparing v-qPCR viable cells estimation with conventional qPCR and plate counting. When cells were treated with peptide conjugates derived from BP100, the observed differences between methods suggested that, in addition to cell death due to the lytic effect of the peptides, there was an induction of the viable but non-culturable state in cells. Notably, a contact test coupled to v-qPCR allowed fast and accurate screening of antimicrobial peptides, and led to the identification of new peptide conjugates active against X. fastidiosa. Conclusions: Antimicrobial peptides active against X. fastidiosa have been identified using an optimized methodology that quantifies viable cells without a cultivation stage, avoiding underestimation or false negative detection of the pathogen due to the viable but non-culturable state, and overestimation of the viable population observed using qPCR. These findings provide new alternative compounds for being tested in planta for the control of X. fastidiosa, and a methodology that enables the fast screening of a large amount of antimicrobials against this plant pathogenic bacterium. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
7. Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method.
- Author
-
Daranas, Núria, Bonaterra, Anna, Francés, Jesús, Cabrefiga, Jordi, Montesinos, Emilio, and Badosa, Esther
- Subjects
- *
POLYMERASE chain reaction , *LACTOBACILLUS plantarum , *DNA , *NUCLEIC acids , *HUMIDITY - Abstract
A viability quantitative PCR (v-qPCR) assay was developed for the unambiguous detection and quantification of Lactobacillus plantarum PM411 viable cells in aerial plant surfaces. A 972-bp region of a PM411 predicted prophage with mosaic architecture enabled the identification of a PM411 strain-specific molecular marker. Three primer sets with different amplicon lengths (92, 188, and 317 bp) and one TaqMan probe were designed. All the qPCR assays showed good linearity over a 4-log range and good efficiencies but differed in sensitivity. The nucleic acid-binding dye PEMAX was used to selectively detect and enumerate viable bacteria by v-qPCR. The primer set amplifying a 188-bp DNA fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed on apple blossoms, pear, strawberry, and kiwifruit leaves in potted plants under controlled environmental conditions, as well as pear and apple blossoms under field conditions, by comparing v-qPCR population estimations to those obtained by qPCR and specific plate counting on de Man-Rogosa-Sharpe (MRS)-rifampin. The population estimation did not differ significantly between methods when conditions were conducive to bacterial survival. However, under stressful conditions, differences between methods were observed due to cell death or viable-but-nonculturable state induction. While qPCR overestimated the population level, plate counting underestimated this value in comparison to v-qPCR. PM411 attained stable population levels of viable cells on the flower environment under high relative humidity. However, the unfavorable conditions on the leaf surface and the relatively dryness in the field caused an important decrease in the viable population. IMPORTANCE The v-qPCR method in combination with plate counting and qPCR is a powerful tool for studies of colonization and survival under field conditions, to improve formulations and delivery strategies of PM411, and to optimize the dose and timing of spray schedules. It is expected that PEMAX v-qPCR could also be developed for monitoring other strains on plant surfaces not only as biological control agents but also beneficial bacteria useful in the sustainable management of crop production. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
8. Experimental basis for the control of plant diseases caused by Xylella fastidiosa using synthetic antimicrobial peptides
- Author
-
Baró Sabé, Aina, Montesinos Seguí, Emilio, Badosa Romañó, Esther, Montesinos Barreda, Laura, Universitat de Girona. Departament d'Enginyeria Química, Agrària i Tecnologia Agroalimentària, and Universitat de Girona. Institut de Tecnologia Agroalimentària
- Subjects
Xylella fastidiosa ,Plant diseases control ,Almond leaf scorch ,Péptidos antimicrobianos ,Almendros ,574 - Ecologia general i biodiversitat ,Control de malalties de les plantes ,632 - Malalties i protecció de les plantes ,Control de enfermedades de las plantas ,Bacterias fitopatógenas ,Viability-qPCR ,Quemadura de la hoja de almendro ,Pèptids antimicrobians ,Antimicrobial peptides ,Socarrat de la fulla d'ametller ,Ametllers ,Bacteris fitopatògens ,Phytopathogenic bacteria - Abstract
Xylella fastidiosa is one of the most damaging phytopathogenic bacteria in the world, causing a variety of diseases with a major economic impact on agriculture and the environment. Until now, no therapeutic solution has been found for its control in affected plants and trees. Antimicrobial peptides (AMPs) represent a promising alternative to traditional compounds due to their activity against a wide range of plant pathogens, low toxicity, mode of action, and availability to create sustainable pesticides as well as disease-resistant crops. Thus, the aim of the present Ph.D. thesis was focused on the study of AMPs as a sustainable alternative for the control of diseases caused by X. fastidiosa. In addition to identifying highly bactericidal AMPs, highlighting important parameters of their activity, the aggressiveness of Spanish isolates of X. fastidiosa was studied in order to design efficient treatment guidelines for its control Xylella fastidiosa és un dels bacteris fitopatògens que provoca més danys a nivell mundial, causant una varietat de malalties amb un gran impacte econòmic en l’agricultura i l’entorn. Fins al moment, no s’ha trobat cap solució terapèutica pel seu control en plantes i arbres afectats. Els pèptids antimicrobians (PAMs) representen una alternativa prometedora als compostos tradicionals degut a la seva activitat contra una àmplia gamma de patògens vegetals, baixa toxicitat, mode d’acció, i disponibilitat per crear pesticides sostenibles i cultius resistents a les malalties. Així doncs, l'objectiu de la present tesi doctoral es va centrar en l'estudi dels PAMs com a alternativa sostenible pel control de les malalties causades per X. fastidiosa. A més d'identificar PAMs altament bactericides, posant de manifest paràmetres importants de la seva activitat, es va estudiar l’agressivitat d’aïllats espanyols de X. fastidiosa per poder dissenyar pautes de tractament eficients pel seu control This work was supported by grants from Ministerio de Ciencia, Innovación y Universidades of Spain (RTI Ref. 2018-099410-B-C21 and E-RTA INIA Ref. 2017-00004-C06-03), from the European Union H2020 programme XFACTORS (cod. 727987), and from INTEROLIVO Growers Association (Ref. 041/18). Aina Baró Sabé was recipient of a research grant from the Secretaria d’Universitats i Recerca, Departament d’Economia i Coneixement, Generalitat de Catalunya (Ref. 2018 FI B00334)
- Published
- 2021
9. Monitoring viable cells of the biological control agent Lactobacillus plantarum PM411 in aerial plant surfaces by means of a strain-specific viability quantitative PCR method
- Author
-
Anna Bonaterra, J. Francés, Jordi Cabrefiga, Emilio Montesinos, Esther Badosa, Núria Daranas, and Ministerio de Economía y Competitividad (Espanya)
- Subjects
0301 basic medicine ,DNA, Bacterial ,030106 microbiology ,Population ,biological control ,Biology ,Real-Time Polymerase Chain Reaction ,Applied Microbiology and Biotechnology ,Fragaria ,Pyrus ,03 medical and health sciences ,chemistry.chemical_compound ,viability-qPCR ,Plant Microbiology ,Species Specificity ,Molecular marker ,TaqMan ,Food science ,education ,Plants -- Diseases and pests ,DNA Primers ,Plant Diseases ,2. Zero hunger ,education.field_of_study ,PEAR ,Microbial Viability ,Ecology ,Bacterial diseases of plants ,Plantes -- Malalties bacterianes ,Amplicon ,Plant Components, Aerial ,Plantes -- Malalties i plagues ,biology.organism_classification ,Pests -- Control ,Plagues -- Control biològic ,chemistry ,Biological Control Agents ,Malus ,Nucleic acid ,Lactobacillus plantarum ,Bacteria ,Food Science ,Biotechnology - Abstract
A viability quantitative PCR (v-qPCR) assay was developed for the unambiguous detection and quantification of Lactobacillus plantarum PM411 viable cells in aerial plant surfaces. A 972-bp region of a PM411 predicted prophage with mosaic architecture enabled the identification of a PM411 strain-specific molecular marker. Three primer sets with different amplicon lengths (92, 188, and 317 bp) and one TaqMan probe were designed. All the qPCR assays showed good linearity over a 4-log range and good efficiencies but differed in sensitivity. The nucleic acid-binding dye PEMAX was used to selectively detect and enumerate viable bacteria by v-qPCR. The primer set amplifying a 188-bp DNA fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed on apple blossoms, pear, strawberry, and kiwifruit leaves in potted plants under controlled environmental conditions, as well as pear and apple blossoms under field conditions, by comparing v-qPCR population estimations to those obtained by qPCR and specific plate counting on de Man-Rogosa-Sharpe (MRS)-rifampin. The population estimation did not differ significantly between methods when conditions were conducive to bacterial survival. However, under stressful conditions, differences between methods were observed due to cell death or viable-but-nonculturable state induction. While qPCR overestimated the population level, plate counting underestimated this value in comparison to v-qPCR. PM411 attained stable population levels of viable cells on the flower environment under high relative humidity. However, the unfavorable conditions on the leaf surface and the relatively dryness in the field caused an important decrease in the viable population. IMPORTANCE The v-qPCR method in combination with plate counting and qPCR is a powerful tool for studies of colonization and survival under field conditions, to improve formulations and delivery strategies of PM411, and to optimize the dose and timing of spray schedules. It is expected that PEMAX v-qPCR could also be developed for monitoring other strains on plant surfaces not only as biological control agents but also beneficial bacteria useful in the sustainable management of crop production.
- Published
- 2020
10. Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the Gardnerella -Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis.
- Author
-
Latka, Agnieszka, Van Simaey, Leen, Reynders, Marijke, Cools, Piet, Rogier, Tess, Lebbe, Barbara, Corsini, Lorenzo, Landlinger, Christine, and Vaneechoutte, Mario
- Subjects
PROPIDIUM monoazide ,BACTERIAL vaginitis ,GENE amplification ,BIOFILMS - Abstract
Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
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