Comparison of three methods for extraction of Mycobacterium avium subspecies paratuberculosis DNA for polymerase chain reaction from broth-based culture systems
Correspondence: 1 Corresponding Author: Ogi Okwumabua, University of Wisconsin, WVDL, 445 Easterday Lane, Madison, WI 53706, e-mail: ogi.okwumabua{at}wvdl.wisc.edu Conventional and real-time polymerase chain reaction (PCR) assays were used to measure the recovery of DNA from Mycobacterium avium subs...
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| Veröffentlicht in: | Journal of veterinary diagnostic investigation 2010, Vol.22 (1), p.67-69 |
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| creator | Okwumabua, Ogi Shull, Eileen O'Connor, Mike Moua, Tou Vue Danz, Tonya Strelow, Kathy |
| description | Correspondence: 1 Corresponding Author: Ogi Okwumabua, University of Wisconsin, WVDL, 445 Easterday Lane, Madison, WI 53706, e-mail: ogi.okwumabua{at}wvdl.wisc.edu
Conventional and real-time polymerase chain reaction (PCR) assays were used to measure the recovery of DNA from Mycobacterium avium subspecies paratuberculosis (MAP) extracted with 3 different methods (MagMAX TM , DNeasy®, and phenol-chloroform) after growth in a broth-based culture system. Of the 304 samples tested, bacterial DNA was detected in 197 (65%) of samples after MagMAX, 156 (51%) after phenol-chloroform, and 123 (40%) after DNeasy extractions. By acid-fast stain, 177 (58%) of the samples yielded acid-fast–positive bacilli, of which 4 were PCR negative by the 3 extraction methods. The results demonstrated that the amplifiable MAP DNA, as evidenced by the number of PCR-positive cultures and amplicon intensity on ethidium bromide–stained agarose gel, was best for MagMAX, intermediate for phenol-chloroform, and least for DNeasy. When subjected to real-time polymerase chain reaction, the MagMAX extracts produced the best results, thereby making it an excellent kit for the efficient extraction of MAP DNA from the broth-based culture system.
Key Words: Broth-based culture DNA extraction Mycobacterium avium subspecies paratuberculosis polymerase chain reaction |
| doi_str_mv | 10.1177/104063871002200111 |
| format | Article |
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Conventional and real-time polymerase chain reaction (PCR) assays were used to measure the recovery of DNA from Mycobacterium avium subspecies paratuberculosis (MAP) extracted with 3 different methods (MagMAX TM , DNeasy®, and phenol-chloroform) after growth in a broth-based culture system. Of the 304 samples tested, bacterial DNA was detected in 197 (65%) of samples after MagMAX, 156 (51%) after phenol-chloroform, and 123 (40%) after DNeasy extractions. By acid-fast stain, 177 (58%) of the samples yielded acid-fast–positive bacilli, of which 4 were PCR negative by the 3 extraction methods. The results demonstrated that the amplifiable MAP DNA, as evidenced by the number of PCR-positive cultures and amplicon intensity on ethidium bromide–stained agarose gel, was best for MagMAX, intermediate for phenol-chloroform, and least for DNeasy. When subjected to real-time polymerase chain reaction, the MagMAX extracts produced the best results, thereby making it an excellent kit for the efficient extraction of MAP DNA from the broth-based culture system.
Key Words: Broth-based culture DNA extraction Mycobacterium avium subspecies paratuberculosis polymerase chain reaction</description><identifier>ISSN: 1040-6387</identifier><identifier>EISSN: 1943-4936</identifier><identifier>DOI: 10.1177/104063871002200111</identifier><identifier>PMID: 20093685</identifier><language>eng</language><publisher>Los Angeles, CA: J Vet Diagn Invest</publisher><subject>accuracy ; Animals ; Bacteriological Techniques ; Cattle ; Cattle Diseases - diagnosis ; Cattle Diseases - microbiology ; cultured cells ; DNA extraction ; DNA extracts ; DNA, Bacterial - chemistry ; DNA, Bacterial - genetics ; laboratory techniques ; methodology ; microbial genetics ; molecular sequence data ; Mycobacterium avium subsp. paratuberculosis ; Mycobacterium avium subsp. paratuberculosis - genetics ; Mycobacterium avium subsp. paratuberculosis - isolation & purification ; nucleotide sequences ; Paratuberculosis - diagnosis ; polymerase chain reaction ; Polymerase Chain Reaction - veterinary ; pretreatment ; Sensitivity and Specificity</subject><ispartof>Journal of veterinary diagnostic investigation, 2010, Vol.22 (1), p.67-69</ispartof><rights>2010 American Association of Veterinary Laboratory Diagnosticians</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c440t-c6c4c246a19de982f15c1a17d50ee09aa6281a804a142d0581771d24400709193</citedby><cites>FETCH-LOGICAL-c440t-c6c4c246a19de982f15c1a17d50ee09aa6281a804a142d0581771d24400709193</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://journals.sagepub.com/doi/pdf/10.1177/104063871002200111$$EPDF$$P50$$Gsage$$H</linktopdf><linktohtml>$$Uhttps://journals.sagepub.com/doi/10.1177/104063871002200111$$EHTML$$P50$$Gsage$$H</linktohtml><link.rule.ids>314,780,784,4022,21817,27921,27922,27923,43619,43620</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20093685$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Okwumabua, Ogi</creatorcontrib><creatorcontrib>Shull, Eileen</creatorcontrib><creatorcontrib>O'Connor, Mike</creatorcontrib><creatorcontrib>Moua, Tou Vue</creatorcontrib><creatorcontrib>Danz, Tonya</creatorcontrib><creatorcontrib>Strelow, Kathy</creatorcontrib><title>Comparison of three methods for extraction of Mycobacterium avium subspecies paratuberculosis DNA for polymerase chain reaction from broth-based culture systems</title><title>Journal of veterinary diagnostic investigation</title><addtitle>J Vet Diagn Invest</addtitle><description>Correspondence: 1 Corresponding Author: Ogi Okwumabua, University of Wisconsin, WVDL, 445 Easterday Lane, Madison, WI 53706, e-mail: ogi.okwumabua{at}wvdl.wisc.edu
Conventional and real-time polymerase chain reaction (PCR) assays were used to measure the recovery of DNA from Mycobacterium avium subspecies paratuberculosis (MAP) extracted with 3 different methods (MagMAX TM , DNeasy®, and phenol-chloroform) after growth in a broth-based culture system. Of the 304 samples tested, bacterial DNA was detected in 197 (65%) of samples after MagMAX, 156 (51%) after phenol-chloroform, and 123 (40%) after DNeasy extractions. By acid-fast stain, 177 (58%) of the samples yielded acid-fast–positive bacilli, of which 4 were PCR negative by the 3 extraction methods. The results demonstrated that the amplifiable MAP DNA, as evidenced by the number of PCR-positive cultures and amplicon intensity on ethidium bromide–stained agarose gel, was best for MagMAX, intermediate for phenol-chloroform, and least for DNeasy. When subjected to real-time polymerase chain reaction, the MagMAX extracts produced the best results, thereby making it an excellent kit for the efficient extraction of MAP DNA from the broth-based culture system.
Key Words: Broth-based culture DNA extraction Mycobacterium avium subspecies paratuberculosis polymerase chain reaction</description><subject>accuracy</subject><subject>Animals</subject><subject>Bacteriological Techniques</subject><subject>Cattle</subject><subject>Cattle Diseases - diagnosis</subject><subject>Cattle Diseases - microbiology</subject><subject>cultured cells</subject><subject>DNA extraction</subject><subject>DNA extracts</subject><subject>DNA, Bacterial - chemistry</subject><subject>DNA, Bacterial - genetics</subject><subject>laboratory techniques</subject><subject>methodology</subject><subject>microbial genetics</subject><subject>molecular sequence data</subject><subject>Mycobacterium avium subsp. paratuberculosis</subject><subject>Mycobacterium avium subsp. paratuberculosis - genetics</subject><subject>Mycobacterium avium subsp. paratuberculosis - isolation & purification</subject><subject>nucleotide sequences</subject><subject>Paratuberculosis - diagnosis</subject><subject>polymerase chain reaction</subject><subject>Polymerase Chain Reaction - veterinary</subject><subject>pretreatment</subject><subject>Sensitivity and Specificity</subject><issn>1040-6387</issn><issn>1943-4936</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9ks1u1DAUhSMEoqXwAizAG9RVqK_j_C2rgQJSgQV0bTnOzcSjeBx8ncK8DY-KhwxskNj4R_c759o-zrLnwF8D1PUVcMmroqmBcyE4B4AH2Tm0sshlW1QP0zoB-ZE4y54Q7TgvRVnD4-ws0YloyvPs58a7WQdLfs_8wOIYEJnDOPqe2OADwx8xaBPtWv94ML5LWwx2cUzfH0daOprRWCSWnHRcOgxmmTxZYm8-Xf92mf10cBg0ITOjtnsW8GQ6BO9YF3wc8y6Ve5akcQnI6EARHT3NHg16Inx2mi-yu5u3Xzfv89vP7z5srm9zIyWPuamMNEJWGtoe20YMUBrQUPclR-St1pVoQDdcapCi52WTHhB6kbS85i20xUV2ufrOwX9bkKJylgxOk96jX0jVRZHARtaJFCtpgicKOKg5WKfDQQFXx2DUv8Ek0YuT_dI57P9K_iSRgKsVIL1FtfNL2Kfr_t_y1aoY7Xb8bgMqcnqaUgOhdve9FUKBqo7nfblyg_ZKb1PW6u6L4FBwaASU6a_8At-QrtU</recordid><startdate>2010</startdate><enddate>2010</enddate><creator>Okwumabua, Ogi</creator><creator>Shull, Eileen</creator><creator>O'Connor, Mike</creator><creator>Moua, Tou Vue</creator><creator>Danz, Tonya</creator><creator>Strelow, Kathy</creator><general>J Vet Diagn Invest</general><general>SAGE Publications</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>2010</creationdate><title>Comparison of three methods for extraction of Mycobacterium avium subspecies paratuberculosis DNA for polymerase chain reaction from broth-based culture systems</title><author>Okwumabua, Ogi ; Shull, Eileen ; O'Connor, Mike ; Moua, Tou Vue ; Danz, Tonya ; Strelow, Kathy</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c440t-c6c4c246a19de982f15c1a17d50ee09aa6281a804a142d0581771d24400709193</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>accuracy</topic><topic>Animals</topic><topic>Bacteriological Techniques</topic><topic>Cattle</topic><topic>Cattle Diseases - diagnosis</topic><topic>Cattle Diseases - microbiology</topic><topic>cultured cells</topic><topic>DNA extraction</topic><topic>DNA extracts</topic><topic>DNA, Bacterial - chemistry</topic><topic>DNA, Bacterial - genetics</topic><topic>laboratory techniques</topic><topic>methodology</topic><topic>microbial genetics</topic><topic>molecular sequence data</topic><topic>Mycobacterium avium subsp. paratuberculosis</topic><topic>Mycobacterium avium subsp. paratuberculosis - genetics</topic><topic>Mycobacterium avium subsp. paratuberculosis - isolation & purification</topic><topic>nucleotide sequences</topic><topic>Paratuberculosis - diagnosis</topic><topic>polymerase chain reaction</topic><topic>Polymerase Chain Reaction - veterinary</topic><topic>pretreatment</topic><topic>Sensitivity and Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Okwumabua, Ogi</creatorcontrib><creatorcontrib>Shull, Eileen</creatorcontrib><creatorcontrib>O'Connor, Mike</creatorcontrib><creatorcontrib>Moua, Tou Vue</creatorcontrib><creatorcontrib>Danz, Tonya</creatorcontrib><creatorcontrib>Strelow, Kathy</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of veterinary diagnostic investigation</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Okwumabua, Ogi</au><au>Shull, Eileen</au><au>O'Connor, Mike</au><au>Moua, Tou Vue</au><au>Danz, Tonya</au><au>Strelow, Kathy</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparison of three methods for extraction of Mycobacterium avium subspecies paratuberculosis DNA for polymerase chain reaction from broth-based culture systems</atitle><jtitle>Journal of veterinary diagnostic investigation</jtitle><addtitle>J Vet Diagn Invest</addtitle><date>2010</date><risdate>2010</risdate><volume>22</volume><issue>1</issue><spage>67</spage><epage>69</epage><pages>67-69</pages><issn>1040-6387</issn><eissn>1943-4936</eissn><abstract>Correspondence: 1 Corresponding Author: Ogi Okwumabua, University of Wisconsin, WVDL, 445 Easterday Lane, Madison, WI 53706, e-mail: ogi.okwumabua{at}wvdl.wisc.edu
Conventional and real-time polymerase chain reaction (PCR) assays were used to measure the recovery of DNA from Mycobacterium avium subspecies paratuberculosis (MAP) extracted with 3 different methods (MagMAX TM , DNeasy®, and phenol-chloroform) after growth in a broth-based culture system. Of the 304 samples tested, bacterial DNA was detected in 197 (65%) of samples after MagMAX, 156 (51%) after phenol-chloroform, and 123 (40%) after DNeasy extractions. By acid-fast stain, 177 (58%) of the samples yielded acid-fast–positive bacilli, of which 4 were PCR negative by the 3 extraction methods. The results demonstrated that the amplifiable MAP DNA, as evidenced by the number of PCR-positive cultures and amplicon intensity on ethidium bromide–stained agarose gel, was best for MagMAX, intermediate for phenol-chloroform, and least for DNeasy. When subjected to real-time polymerase chain reaction, the MagMAX extracts produced the best results, thereby making it an excellent kit for the efficient extraction of MAP DNA from the broth-based culture system.
Key Words: Broth-based culture DNA extraction Mycobacterium avium subspecies paratuberculosis polymerase chain reaction</abstract><cop>Los Angeles, CA</cop><pub>J Vet Diagn Invest</pub><pmid>20093685</pmid><doi>10.1177/104063871002200111</doi><tpages>3</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | accuracy Animals Bacteriological Techniques Cattle Cattle Diseases - diagnosis Cattle Diseases - microbiology cultured cells DNA extraction DNA extracts DNA, Bacterial - chemistry DNA, Bacterial - genetics laboratory techniques methodology microbial genetics molecular sequence data Mycobacterium avium subsp. paratuberculosis Mycobacterium avium subsp. paratuberculosis - genetics Mycobacterium avium subsp. paratuberculosis - isolation & purification nucleotide sequences Paratuberculosis - diagnosis polymerase chain reaction Polymerase Chain Reaction - veterinary pretreatment Sensitivity and Specificity |
| title | Comparison of three methods for extraction of Mycobacterium avium subspecies paratuberculosis DNA for polymerase chain reaction from broth-based culture systems |
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