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Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring
Droplet digital polymerase chain reaction (ddPCR) was evaluated for the detection of fecal indicator bacteria (FIB), Enterococcus spp., in San Diego County beach water samples collected under diverse conditions, from multiple pollution sources, as part of regulatory monitoring activities over 20 mon...
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| Published in: | Journal of microbiological methods 2021-05, Vol.184, p.106206, Article 106206 |
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| creator | Crain, Chad Kezer, Keith Steele, Syreeta Owiti, Judith Rao, Sphoorthy Victorio, Maria Austin, Brett Volner, Alon Draper, William Griffith, John Steele, Joshua Seifert, Marva |
| description | Droplet digital polymerase chain reaction (ddPCR) was evaluated for the detection of fecal indicator bacteria (FIB), Enterococcus spp., in San Diego County beach water samples collected under diverse conditions, from multiple pollution sources, as part of regulatory monitoring activities over 20 months. Two US EPA-approved methods, qPCR (EPA 1609.1) and Enterolert (SM9230D), were used as reference comparator methods. A total of 361 samples were assayed by both ddPCR and qPCR and yielded an acceptable Index of Agreement (IA) of 0.89, based on EPA Site-Specific analysis guidelines. A Pearson's correlation coefficient of r = 0.87 (p |
| doi_str_mv | 10.1016/j.mimet.2021.106206 |
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•Detection of Enterococcus spp. 23S rDNA closely aligns with MPN for measuring FIB exceedances.•Intrinsic copy number equation (ICE) scales ddPCR results for comparison to MPN values.•ddPCR and qPCR results highly correlate for detecting Enterococcus spp. 23S rDNA.</description><identifier>ISSN: 0167-7012</identifier><identifier>EISSN: 1872-8359</identifier><identifier>DOI: 10.1016/j.mimet.2021.106206</identifier><identifier>PMID: 33766607</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>AB411 ; Bacteria - genetics ; Bacteria - growth & development ; Bacteria - isolation & purification ; Droplet digital polymerase chain reaction ; Enterococci ; Environmental Monitoring - methods ; Feces - microbiology ; Humans ; Intrinsic copy number equation ; Quantitative polymerase chain reaction ; Real-Time Polymerase Chain Reaction - methods ; Seawater - chemistry ; Seawater - microbiology ; United State Environmental Protection Agency ; United States ; United States Environmental Protection Agency ; Water Pollution - analysis ; Water Quality</subject><ispartof>Journal of microbiological methods, 2021-05, Vol.184, p.106206, Article 106206</ispartof><rights>2021</rights><rights>Published by Elsevier B.V.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c359t-2b9574b3518dcbc7554d388e187838b8bb42daad7fcd7e0801c51c09487c63c13</citedby><cites>FETCH-LOGICAL-c359t-2b9574b3518dcbc7554d388e187838b8bb42daad7fcd7e0801c51c09487c63c13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33766607$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Crain, Chad</creatorcontrib><creatorcontrib>Kezer, Keith</creatorcontrib><creatorcontrib>Steele, Syreeta</creatorcontrib><creatorcontrib>Owiti, Judith</creatorcontrib><creatorcontrib>Rao, Sphoorthy</creatorcontrib><creatorcontrib>Victorio, Maria</creatorcontrib><creatorcontrib>Austin, Brett</creatorcontrib><creatorcontrib>Volner, Alon</creatorcontrib><creatorcontrib>Draper, William</creatorcontrib><creatorcontrib>Griffith, John</creatorcontrib><creatorcontrib>Steele, Joshua</creatorcontrib><creatorcontrib>Seifert, Marva</creatorcontrib><title>Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring</title><title>Journal of microbiological methods</title><addtitle>J Microbiol Methods</addtitle><description>Droplet digital polymerase chain reaction (ddPCR) was evaluated for the detection of fecal indicator bacteria (FIB), Enterococcus spp., in San Diego County beach water samples collected under diverse conditions, from multiple pollution sources, as part of regulatory monitoring activities over 20 months. Two US EPA-approved methods, qPCR (EPA 1609.1) and Enterolert (SM9230D), were used as reference comparator methods. A total of 361 samples were assayed by both ddPCR and qPCR and yielded an acceptable Index of Agreement (IA) of 0.89, based on EPA Site-Specific analysis guidelines. A Pearson's correlation coefficient of r = 0.87 (p < 0.001), further indicated a strong relationship between the methods results. From the 361 samples, 185 split samples with ddPCR and Enterolert values within the limits of quantification, were used as a ‘training’ data set to derive an intrinsic copy number equation (ICE) for scaling ddPCR gene copy number to Enterolert most probable number (MPN). Of the 1993 samples that comprised the complete ‘test’ data set assayed by ddPCR and Enterolert, 1086 generated results that fell within the limits of quantification for Enterolert and yielded an overall IA of 0.64. Re-analysis using median as a measure of central tendency to account for significant skewing of Enterolert data yielded an IA of 0.72. Beach grouping-specific IA values ranged from 0.63 to 0.93. Pearson's correlation coefficient, r, ranged from 0.13 to 0.94 within beach groupings and generated a combined value of 0.60 for all groupings. Using the ICE, a ddPCR advisory threshold of 1413 DNA copy number/100 mL was empirically determined to be the equivalent to the California Enterolert beach action threshold of 104 MPN/100 mL, based on comparison with all 1993 paired ddPCR and Enterolert results. Using the 1413 DNA copy number/100 mL as a beach action threshold for ddPCR resulted in a 90.4% agreement with Enterolert (6.0% false negative and 3.7% false positive). Together these findings support the conclusion that ddPCR readouts align closely with Enterolert MPN for identifying FIB exceedance levels of Enterococcus spp. in coastal waters of San Diego, CA.
•Detection of Enterococcus spp. 23S rDNA closely aligns with MPN for measuring FIB exceedances.•Intrinsic copy number equation (ICE) scales ddPCR results for comparison to MPN values.•ddPCR and qPCR results highly correlate for detecting Enterococcus spp. 23S rDNA.</description><subject>AB411</subject><subject>Bacteria - genetics</subject><subject>Bacteria - growth & development</subject><subject>Bacteria - isolation & purification</subject><subject>Droplet digital polymerase chain reaction</subject><subject>Enterococci</subject><subject>Environmental Monitoring - methods</subject><subject>Feces - microbiology</subject><subject>Humans</subject><subject>Intrinsic copy number equation</subject><subject>Quantitative polymerase chain reaction</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Seawater - chemistry</subject><subject>Seawater - microbiology</subject><subject>United State Environmental Protection Agency</subject><subject>United States</subject><subject>United States Environmental Protection Agency</subject><subject>Water Pollution - analysis</subject><subject>Water Quality</subject><issn>0167-7012</issn><issn>1872-8359</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><recordid>eNp9kE1OwzAQhS0EoqVwAiTkCyTYcRK7CxZVVX6kSiAEKxaWM3aQqyQOdgrq7XEJZclqpJn3Zt58CF1SklJCy-tN2trWDGlGMho7ZUbKIzSlgmeJYMX8GE2jiiec0GyCzkLYEEILlotTNGGMl2VJ-BS9Lfq-saAG6zrsaqz10_IZ185jbQYDh_aqG4x34AC2AYe-T7HtMDgVBtXgLxWH-GOrGjvscOs6Ozhvu_dzdFKrJpiL3zpDr7erl-V9sn68e1gu1gnEmEOSVfOC5xUrqNBQAS-KXDMhTPxEMFGJqsozrZTmNWhuiCAUCgpkngsOJQPKZoiNe8G7ELypZe9tq_xOUiL3qORG_qCSe1RyRBVdV6Or31at0X-eA5souBkFJmb_tMbLANZ0YLT1kYzUzv574BuzJ3vd</recordid><startdate>202105</startdate><enddate>202105</enddate><creator>Crain, Chad</creator><creator>Kezer, Keith</creator><creator>Steele, Syreeta</creator><creator>Owiti, Judith</creator><creator>Rao, Sphoorthy</creator><creator>Victorio, Maria</creator><creator>Austin, Brett</creator><creator>Volner, Alon</creator><creator>Draper, William</creator><creator>Griffith, John</creator><creator>Steele, Joshua</creator><creator>Seifert, Marva</creator><general>Elsevier B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope></search><sort><creationdate>202105</creationdate><title>Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring</title><author>Crain, Chad ; Kezer, Keith ; Steele, Syreeta ; Owiti, Judith ; Rao, Sphoorthy ; Victorio, Maria ; Austin, Brett ; Volner, Alon ; Draper, William ; Griffith, John ; Steele, Joshua ; Seifert, Marva</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c359t-2b9574b3518dcbc7554d388e187838b8bb42daad7fcd7e0801c51c09487c63c13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>AB411</topic><topic>Bacteria - genetics</topic><topic>Bacteria - growth & development</topic><topic>Bacteria - isolation & purification</topic><topic>Droplet digital polymerase chain reaction</topic><topic>Enterococci</topic><topic>Environmental Monitoring - methods</topic><topic>Feces - microbiology</topic><topic>Humans</topic><topic>Intrinsic copy number equation</topic><topic>Quantitative polymerase chain reaction</topic><topic>Real-Time Polymerase Chain Reaction - methods</topic><topic>Seawater - chemistry</topic><topic>Seawater - microbiology</topic><topic>United State Environmental Protection Agency</topic><topic>United States</topic><topic>United States Environmental Protection Agency</topic><topic>Water Pollution - analysis</topic><topic>Water Quality</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Crain, Chad</creatorcontrib><creatorcontrib>Kezer, Keith</creatorcontrib><creatorcontrib>Steele, Syreeta</creatorcontrib><creatorcontrib>Owiti, Judith</creatorcontrib><creatorcontrib>Rao, Sphoorthy</creatorcontrib><creatorcontrib>Victorio, Maria</creatorcontrib><creatorcontrib>Austin, Brett</creatorcontrib><creatorcontrib>Volner, Alon</creatorcontrib><creatorcontrib>Draper, William</creatorcontrib><creatorcontrib>Griffith, John</creatorcontrib><creatorcontrib>Steele, Joshua</creatorcontrib><creatorcontrib>Seifert, Marva</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>Journal of microbiological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Crain, Chad</au><au>Kezer, Keith</au><au>Steele, Syreeta</au><au>Owiti, Judith</au><au>Rao, Sphoorthy</au><au>Victorio, Maria</au><au>Austin, Brett</au><au>Volner, Alon</au><au>Draper, William</au><au>Griffith, John</au><au>Steele, Joshua</au><au>Seifert, Marva</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring</atitle><jtitle>Journal of microbiological methods</jtitle><addtitle>J Microbiol Methods</addtitle><date>2021-05</date><risdate>2021</risdate><volume>184</volume><spage>106206</spage><pages>106206-</pages><artnum>106206</artnum><issn>0167-7012</issn><eissn>1872-8359</eissn><abstract>Droplet digital polymerase chain reaction (ddPCR) was evaluated for the detection of fecal indicator bacteria (FIB), Enterococcus spp., in San Diego County beach water samples collected under diverse conditions, from multiple pollution sources, as part of regulatory monitoring activities over 20 months. Two US EPA-approved methods, qPCR (EPA 1609.1) and Enterolert (SM9230D), were used as reference comparator methods. A total of 361 samples were assayed by both ddPCR and qPCR and yielded an acceptable Index of Agreement (IA) of 0.89, based on EPA Site-Specific analysis guidelines. A Pearson's correlation coefficient of r = 0.87 (p < 0.001), further indicated a strong relationship between the methods results. From the 361 samples, 185 split samples with ddPCR and Enterolert values within the limits of quantification, were used as a ‘training’ data set to derive an intrinsic copy number equation (ICE) for scaling ddPCR gene copy number to Enterolert most probable number (MPN). Of the 1993 samples that comprised the complete ‘test’ data set assayed by ddPCR and Enterolert, 1086 generated results that fell within the limits of quantification for Enterolert and yielded an overall IA of 0.64. Re-analysis using median as a measure of central tendency to account for significant skewing of Enterolert data yielded an IA of 0.72. Beach grouping-specific IA values ranged from 0.63 to 0.93. Pearson's correlation coefficient, r, ranged from 0.13 to 0.94 within beach groupings and generated a combined value of 0.60 for all groupings. Using the ICE, a ddPCR advisory threshold of 1413 DNA copy number/100 mL was empirically determined to be the equivalent to the California Enterolert beach action threshold of 104 MPN/100 mL, based on comparison with all 1993 paired ddPCR and Enterolert results. Using the 1413 DNA copy number/100 mL as a beach action threshold for ddPCR resulted in a 90.4% agreement with Enterolert (6.0% false negative and 3.7% false positive). Together these findings support the conclusion that ddPCR readouts align closely with Enterolert MPN for identifying FIB exceedance levels of Enterococcus spp. in coastal waters of San Diego, CA.
•Detection of Enterococcus spp. 23S rDNA closely aligns with MPN for measuring FIB exceedances.•Intrinsic copy number equation (ICE) scales ddPCR results for comparison to MPN values.•ddPCR and qPCR results highly correlate for detecting Enterococcus spp. 23S rDNA.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>33766607</pmid><doi>10.1016/j.mimet.2021.106206</doi></addata></record> |
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| source | ScienceDirect Journals |
| subjects | AB411 Bacteria - genetics Bacteria - growth & development Bacteria - isolation & purification Droplet digital polymerase chain reaction Enterococci Environmental Monitoring - methods Feces - microbiology Humans Intrinsic copy number equation Quantitative polymerase chain reaction Real-Time Polymerase Chain Reaction - methods Seawater - chemistry Seawater - microbiology United State Environmental Protection Agency United States United States Environmental Protection Agency Water Pollution - analysis Water Quality |
| title | Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring |
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