Development of PMA real-time PCR method to quantify viable cells of Pantoea agglomerans CPA-2, an antagonist to control the major postharvest diseases on oranges.
Identifieur interne : 000B37 ( Main/Exploration ); précédent : 000B36; suivant : 000B38Development of PMA real-time PCR method to quantify viable cells of Pantoea agglomerans CPA-2, an antagonist to control the major postharvest diseases on oranges.
Auteurs : Lourdes Soto-Mu Oz [Espagne] ; Neus Teixid [Espagne] ; Josep Usall [Espagne] ; Inmaculada Vi As [Espagne] ; Ana Crespo-Sempere [Espagne] ; Rosario Torres [Espagne]Source :
- International journal of food microbiology [ 1879-3460 ] ; 2014.
English descriptors
- KwdEn :
- MESH :
- chemical , analogs & derivatives : Propidium.
- chemical , chemistry : Azides, Propidium.
- chemical : Biological Control Agents.
- genetics : Pantoea.
- methods : Food Microbiology.
- microbiology : Citrus sinensis, Fruit.
- physiology : Pantoea.
- Microbial Viability, Real-Time Polymerase Chain Reaction, Stem Cells.
Abstract
Dilution plating is the quantification method commonly used to estimate the population level of postharvest biocontrol agents, but this method does not permit a distinction among introduced and indigenous strains. Recently, molecular techniques based on DNA amplification such as quantitative real-time PCR (qPCR) have been successfully applied for their high strain-specific detection level. However, the ability of qPCR to distinguish viable and nonviable cells is limited. A promising strategy to avoid this issue relies on the use of nucleic acid intercalating dyes, such as propidium monoazide (PMA), as a sample pretreatment prior to the qPCR. The objective of this study was to optimize a protocol based on PMA pre-treatment samples combined with qPCR to distinguish and quantify viable cells of the biocontrol agent P. agglomerans CPA-2 applied as a postharvest treatment on orange. The efficiency of PMA-qPCR method under the established conditions (30μM PMA for 20min of incubation followed by 30min of LED light exposure) was evaluated on an orange matrix. Results showed no difference in CFU or cells counts of viable cells between PMA-qPCR and dilution plating. Samples of orange matrix inoculated with a mixture of viable/dead cells showed 5.59log10 CFU/ml by dilution plating, 8.25log10 cells/ml by qPCR, and 5.93log10 cells/ml by PMA-qPCR. Furthermore, samples inoculated with heat-killed cells were not detected by dilution plating and PMA-qPCR, while by qPCR was of 8.16log10 cells/ml. The difference in quantification cycles (Cq) among qPCR and PMA-qPCR was approximately 16cycles, which means a reduction of 65,536 fold of the dead cells detected. In conclusion, PMA-qPCR method is a suitable tool for quantify viable CPA-2 cells, which could be useful to estimate the ability of this antagonist to colonize the orange surface.
DOI: 10.1016/j.ijfoodmicro.2014.04.011
PubMed: 24786552
Affiliations:
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Rovira Roure, 191, 25198 Lleida Catalonia, Spain.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>Food Technology Department, Lleida University, XaRTA-Postharvest, Agrotecnio Center, Av. 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Rovira Roure, 191, 25198 Lleida Catalonia, Spain.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>Food Technology Department, Lleida University, XaRTA-Postharvest, Agrotecnio Center, Av. Rovira Roure, 191, 25198 Lleida Catalonia</wicri:regionArea><placeName><region nuts="2" type="communauté">Catalogne</region></placeName></affiliation></author><author><name sortKey="Crespo Sempere, Ana" sort="Crespo Sempere, Ana" uniqKey="Crespo Sempere A" first="Ana" last="Crespo-Sempere">Ana Crespo-Sempere</name><affiliation wicri:level="2"><nlm:affiliation>Applied Mycology Unit, Food Technology Department, Lleida University, XaRTA-UTPV, Agrotecnio Center, Av. Rovira Roure, 191, 25198 Lleida Catalonia, Spain.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>Applied Mycology Unit, Food Technology Department, Lleida University, XaRTA-UTPV, Agrotecnio Center, Av. Rovira Roure, 191, 25198 Lleida Catalonia</wicri:regionArea><placeName><region nuts="2" type="communauté">Catalogne</region></placeName></affiliation></author><author><name sortKey="Torres, Rosario" sort="Torres, Rosario" uniqKey="Torres R" first="Rosario" last="Torres">Rosario Torres</name><affiliation wicri:level="2"><nlm:affiliation>IRTA, XaRTA-Postharvest, Edifici Fruitcentre, Parc Científic i Tecnològic Agroalimentari de Lleida, Parc de Gardeny, 25003 Lleida Catalonia, Spain. Electronic address: rosario.torres@irta.cat.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>IRTA, XaRTA-Postharvest, Edifici Fruitcentre, Parc Científic i Tecnològic Agroalimentari de Lleida, Parc de Gardeny, 25003 Lleida Catalonia</wicri:regionArea><placeName><region nuts="2" type="communauté">Catalogne</region></placeName></affiliation></author></analytic><series><title level="j">International journal of food microbiology</title><idno type="eISSN">1879-3460</idno><imprint><date when="2014" type="published">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Azides (chemistry)</term><term>Biological Control Agents</term><term>Citrus sinensis (microbiology)</term><term>Food Microbiology (methods)</term><term>Fruit (microbiology)</term><term>Microbial Viability</term><term>Pantoea (genetics)</term><term>Pantoea (physiology)</term><term>Propidium (analogs & derivatives)</term><term>Propidium (chemistry)</term><term>Real-Time Polymerase Chain Reaction</term><term>Stem Cells</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analogs & derivatives" xml:lang="en"><term>Propidium</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Azides</term><term>Propidium</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Biological Control Agents</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Pantoea</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Food Microbiology</term></keywords><keywords scheme="MESH" qualifier="microbiology" xml:lang="en"><term>Citrus sinensis</term><term>Fruit</term></keywords><keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>Pantoea</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Microbial Viability</term><term>Real-Time Polymerase Chain Reaction</term><term>Stem Cells</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Dilution plating is the quantification method commonly used to estimate the population level of postharvest biocontrol agents, but this method does not permit a distinction among introduced and indigenous strains. Recently, molecular techniques based on DNA amplification such as quantitative real-time PCR (qPCR) have been successfully applied for their high strain-specific detection level. However, the ability of qPCR to distinguish viable and nonviable cells is limited. A promising strategy to avoid this issue relies on the use of nucleic acid intercalating dyes, such as propidium monoazide (PMA), as a sample pretreatment prior to the qPCR. The objective of this study was to optimize a protocol based on PMA pre-treatment samples combined with qPCR to distinguish and quantify viable cells of the biocontrol agent P. agglomerans CPA-2 applied as a postharvest treatment on orange. The efficiency of PMA-qPCR method under the established conditions (30μM PMA for 20min of incubation followed by 30min of LED light exposure) was evaluated on an orange matrix. Results showed no difference in CFU or cells counts of viable cells between PMA-qPCR and dilution plating. Samples of orange matrix inoculated with a mixture of viable/dead cells showed 5.59log10 CFU/ml by dilution plating, 8.25log10 cells/ml by qPCR, and 5.93log10 cells/ml by PMA-qPCR. Furthermore, samples inoculated with heat-killed cells were not detected by dilution plating and PMA-qPCR, while by qPCR was of 8.16log10 cells/ml. The difference in quantification cycles (Cq) among qPCR and PMA-qPCR was approximately 16cycles, which means a reduction of 65,536 fold of the dead cells detected. In conclusion, PMA-qPCR method is a suitable tool for quantify viable CPA-2 cells, which could be useful to estimate the ability of this antagonist to colonize the orange surface.</div></front></TEI><affiliations><list><country><li>Espagne</li></country><region><li>Catalogne</li></region></list><tree><country name="Espagne"><region name="Catalogne"><name sortKey="Soto Mu Oz, Lourdes" sort="Soto Mu Oz, Lourdes" uniqKey="Soto Mu Oz L" first="Lourdes" last="Soto-Mu Oz">Lourdes Soto-Mu Oz</name></region><name sortKey="Crespo Sempere, Ana" sort="Crespo Sempere, Ana" uniqKey="Crespo Sempere A" first="Ana" last="Crespo-Sempere">Ana Crespo-Sempere</name><name sortKey="Teixid, Neus" sort="Teixid, Neus" uniqKey="Teixid N" first="Neus" last="Teixid">Neus Teixid</name><name sortKey="Torres, Rosario" sort="Torres, Rosario" uniqKey="Torres R" first="Rosario" last="Torres">Rosario Torres</name><name sortKey="Usall, Josep" sort="Usall, Josep" uniqKey="Usall J" first="Josep" last="Usall">Josep Usall</name><name sortKey="Vi As, Inmaculada" sort="Vi As, Inmaculada" uniqKey="Vi As I" first="Inmaculada" last="Vi As">Inmaculada Vi As</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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