Enzymatic Treatment of Specimens before DNA Extraction Directly Influences Molecular Detection of Infectious Agents
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{"title"=>"Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious agents", "type"=>"journal", "authors"=>[{"first_name"=>"Pablo", "last_name"=>"Goldschmidt", "scopus_author_id"=>"7006736454"}, {"first_name"=>"Sandrine", "last_name"=>"Degorge", "scopus_author_id"=>"23469196800"}, {"first_name"=>"Lilia", "last_name"=>"Merabet", "scopus_author_id"=>"57197075373"}, {"first_name"=>"Christine", "last_name"=>"Chaumeil", "scopus_author_id"=>"6603893346"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84903287067", "doi"=>"10.1371/journal.pone.0094886", "issn"=>"19326203", "pui"=>"373396652", "isbn"=>"0933-7407", "pmid"=>"24936792", "scopus"=>"2-s2.0-84903287067"}, "id"=>"ef5f176b-cd28-3d11-81f7-60275742a32f", "abstract"=>"INTRODUCTION: Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification.\\n\\nMETHODS: Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis).\\n\\nRESULTS: Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification.\\n\\nDISCUSSION: Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis.", "link"=>"http://www.mendeley.com/research/enzymatic-treatment-specimens-before-dna-extraction-directly-influences-molecular-detection-infectio", "reader_count"=>31, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Researcher"=>6, "Student > Ph. D. Student"=>9, "Student > Postgraduate"=>1, "Other"=>1, "Student > Master"=>7, "Student > Bachelor"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Researcher"=>6, "Student > Ph. D. Student"=>9, "Student > Postgraduate"=>1, "Other"=>1, "Student > Master"=>7, "Student > Bachelor"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>1, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Medicine and Dentistry"=>5, "Agricultural and Biological Sciences"=>15, "Immunology and Microbiology"=>4, "Earth and Planetary Sciences"=>1, "Economics, Econometrics and Finance"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>4}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Economics, Econometrics and Finance"=>{"Economics, Econometrics and Finance"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>15}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>1}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"Mexico"=>1, "Slovenia"=>1, "India"=>1}, "group_count"=>1}

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  • {"files"=>["https://ndownloader.figshare.com/files/1537275", "https://ndownloader.figshare.com/files/1537276"], "description"=>"<div><p>Introduction</p><p>Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification.</p><p>Methods</p><p><i>Staphylococcus epidermidis</i>, <i>Streptococcus mitis</i>, <i>Propionibacterium acnes</i>, <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, <i>Candida albicans</i>, <i>Aspergillus niger and Fusarium solani</i> were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid <i>Herpesvirus</i>. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous <i>Fungi</i> with HRM (real-time PCR followed by high-resolution melting analysis).</p><p>Results</p><p>Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for <i>Fungi</i> and Proteinase K for <i>Bacteria</i>. The addition of lyticase and proteinase K did not improve results. For <i>Fungi</i> the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification.</p><p>Discussion</p><p>Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis.</p></div>", "links"=>[], "tags"=>["biotechnology", "cell biology", "Molecular cell biology", "microbiology", "Veterinary science", "Veterinary microbiology", "Diagnostic medicine", "epidemiology", "Infectious diseases", "specimens", "dna", "extraction", "influences", "molecular", "detection", "infectious"], "article_id"=>1059449, "categories"=>["Biological Sciences"], "users"=>["Pablo Goldschmidt", "Sandrine Degorge", "Lilia Merabet", "Christine Chaumeil"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0094886.s001", "https://dx.doi.org/10.1371/journal.pone.0094886.s002"], "stats"=>{"downloads"=>3, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Enzymatic_Treatment_of_Specimens_before_DNA_Extraction_Directly_Influences_Molecular_Detection_of_Infectious_Agents_/1059449", "title"=>"Enzymatic Treatment of Specimens before DNA Extraction Directly Influences Molecular Detection of Infectious Agents", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-06-17 02:49:36"}

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Relative Metric

{"start_date"=>"2014-01-01T00:00:00Z", "end_date"=>"2014-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Molecular biology", "average_usage"=>[292, 461]}, {"subject_area"=>"/Biology and life sciences/Organisms", "average_usage"=>[310]}]}
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