Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice.
Identifieur interne : 000A28 ( PubMed/Corpus ); précédent : 000A27; suivant : 000A29Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice.
Auteurs : Angelique Renard ; Perla G Mez Di Marco ; Marcos Egea-Cortines ; Julia WeissSource :
- International journal of food microbiology [ 0168-1605 ] ; 2008.
English descriptors
- KwdEn :
- Beverages (microbiology), Citrus sinensis (microbiology), Colony Count, Microbial (methods), DNA, Fungal (chemistry), DNA, Fungal (genetics), Food Contamination (analysis), Gene Amplification, Genome, Fungal (genetics), Hanseniaspora (isolation & purification), Polymerase Chain Reaction (methods), RNA, Ribosomal, 5.8S (chemistry), RNA, Ribosomal, 5.8S (genetics), Saccharomyces cerevisiae (isolation & purification), Sensitivity and Specificity, Species Specificity.
- MESH :
- chemical , chemistry : DNA, Fungal, RNA, Ribosomal, 5.8S.
- chemical , genetics : DNA, Fungal, RNA, Ribosomal, 5.8S.
- analysis : Food Contamination.
- genetics : Genome, Fungal.
- isolation & purification : Hanseniaspora, Saccharomyces cerevisiae.
- methods : Colony Count, Microbial, Polymerase Chain Reaction.
- microbiology : Beverages, Citrus sinensis.
- Gene Amplification, Sensitivity and Specificity, Species Specificity.
Abstract
Small cell numbers in complex food matrices and undefined PCR inhibitors often limit detection and identification of DNA species by molecular techniques. Thus in many industrial situations enrichment growths are performed. However, growth speed of different species in complex microbial mixtures in defined media is in most cases different, thus final results do not always reflect the starting situation. We tested DNA-strand displacement whole genome amplification as a possible substitute of enrichment growth. Using whole genome amplification on orange juice contaminated with Saccharomyces cerevisiae, we lowered detection level from 10(6) down to 10(2) cfu/ml. Whole genome amplification showed to be linear (R=0.992) and the relative yeast DNA copy number compared to other DNA templates did not change thus allowing quantitative estimation of initial contamination by quantitative PCR. Using melting point analysis, we were able to distinguish between the PCR products of the 5.8S-ITS region, obtained with universal primers from pure cultures of S. cerevisiae and Hanseniaspora uvarum, two major spoilage yeasts in orange juice and forming part of wine microbiota during fermentation. However, in mixed-contaminated samples, identification of both species was hampered by preferential appearance of the melting peak coinciding with H. uvarum, except when S. cerevisiae was the dominating species. Application of whole genome amplification did not prevent the preferential detection of H. uvarum. This handicap was resolved by applying an enrichment procedure up to saturation after which the melting peak of both species could clearly be identified.
DOI: 10.1016/j.ijfoodmicro.2008.05.021
PubMed: 18597878
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pubmed:18597878Le document en format XML
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Thus in many industrial situations enrichment growths are performed. However, growth speed of different species in complex microbial mixtures in defined media is in most cases different, thus final results do not always reflect the starting situation. We tested DNA-strand displacement whole genome amplification as a possible substitute of enrichment growth. Using whole genome amplification on orange juice contaminated with Saccharomyces cerevisiae, we lowered detection level from 10(6) down to 10(2) cfu/ml. Whole genome amplification showed to be linear (R=0.992) and the relative yeast DNA copy number compared to other DNA templates did not change thus allowing quantitative estimation of initial contamination by quantitative PCR. Using melting point analysis, we were able to distinguish between the PCR products of the 5.8S-ITS region, obtained with universal primers from pure cultures of S. cerevisiae and Hanseniaspora uvarum, two major spoilage yeasts in orange juice and forming part of wine microbiota during fermentation. However, in mixed-contaminated samples, identification of both species was hampered by preferential appearance of the melting peak coinciding with H. uvarum, except when S. cerevisiae was the dominating species. Application of whole genome amplification did not prevent the preferential detection of H. uvarum. This handicap was resolved by applying an enrichment procedure up to saturation after which the melting peak of both species could clearly be identified.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">18597878</PMID><DateCreated><Year>2008</Year><Month>8</Month><Day>4</Day></DateCreated><DateCompleted><Year>2008</Year><Month>12</Month><Day>01</Day></DateCompleted><DateRevised><Year>2008</Year><Month>8</Month><Day>4</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0168-1605</ISSN><JournalIssue CitedMedium="Print"><Volume>126</Volume><Issue>1-2</Issue><PubDate><Year>2008</Year><Month>Aug</Month><Day>15</Day></PubDate></JournalIssue><Title>International journal of food microbiology</Title><ISOAbbreviation>Int. J. Food Microbiol.</ISOAbbreviation></Journal><ArticleTitle>Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice.</ArticleTitle><Pagination><MedlinePgn>195-201</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.ijfoodmicro.2008.05.021</ELocationID><Abstract><AbstractText>Small cell numbers in complex food matrices and undefined PCR inhibitors often limit detection and identification of DNA species by molecular techniques. Thus in many industrial situations enrichment growths are performed. However, growth speed of different species in complex microbial mixtures in defined media is in most cases different, thus final results do not always reflect the starting situation. We tested DNA-strand displacement whole genome amplification as a possible substitute of enrichment growth. Using whole genome amplification on orange juice contaminated with Saccharomyces cerevisiae, we lowered detection level from 10(6) down to 10(2) cfu/ml. Whole genome amplification showed to be linear (R=0.992) and the relative yeast DNA copy number compared to other DNA templates did not change thus allowing quantitative estimation of initial contamination by quantitative PCR. Using melting point analysis, we were able to distinguish between the PCR products of the 5.8S-ITS region, obtained with universal primers from pure cultures of S. cerevisiae and Hanseniaspora uvarum, two major spoilage yeasts in orange juice and forming part of wine microbiota during fermentation. However, in mixed-contaminated samples, identification of both species was hampered by preferential appearance of the melting peak coinciding with H. uvarum, except when S. cerevisiae was the dominating species. Application of whole genome amplification did not prevent the preferential detection of H. uvarum. This handicap was resolved by applying an enrichment procedure up to saturation after which the melting peak of both species could clearly be identified.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Renard</LastName><ForeName>Angelique</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Agricultural Science and Technology Department, Genetics, Institute of Plant Biotechnology, Universidad Politécnica de Cartagena, 30203 Cartagena, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gómez di Marco</LastName><ForeName>Perla</ForeName><Initials>P</Initials></Author><Author ValidYN="Y"><LastName>Egea-Cortines</LastName><ForeName>Marcos</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Weiss</LastName><ForeName>Julia</ForeName><Initials>J</Initials></Author></AuthorList><Language>ENG</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType 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MajorTopicYN="N">microbiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D015169" MajorTopicYN="N">Colony Count, Microbial</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="N">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004271" MajorTopicYN="N">DNA, Fungal</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005506" MajorTopicYN="N">Food Contamination</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="Y">analysis</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005784" MajorTopicYN="N">Gene Amplification</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D016681" MajorTopicYN="N">Genome, Fungal</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D055321" MajorTopicYN="N">Hanseniaspora</DescriptorName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D016133" MajorTopicYN="N">Polymerase Chain Reaction</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D012340" MajorTopicYN="N">RNA, Ribosomal, 5.8S</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D012441" MajorTopicYN="N">Saccharomyces cerevisiae</DescriptorName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D012680" MajorTopicYN="N">Sensitivity and Specificity</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013045" MajorTopicYN="N">Species Specificity</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2007</Year><Month>8</Month><Day>20</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2008</Year><Month>5</Month><Day>16</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2008</Year><Month>5</Month><Day>17</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2008</Year><Month>7</Month><Day>4</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2008</Year><Month>12</Month><Day>17</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2008</Year><Month>7</Month><Day>4</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">18597878</ArticleId><ArticleId IdType="pii">S0168-1605(08)00282-1</ArticleId><ArticleId 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