Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

Alexander Schriewer; Kelly D. Goodwin; Christopher D. Sinigalliano; Annie M. Cox; David Wanless; Jakob Bartkowiak; Darcy L. Ebentier; Kaitlyn T. Hanley; Jared Ervin; Louise A. Deering; Orin C. Shanks; Lindsay A. Peed; Wim G. Meijer; John F. Griffith; Jorge SantoDomingo; Jennifer A. Jay; Patricia A. Holden; Stefan Wuertz

doi:10.1016/j.watres.2013.03.062

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

Alexander Schriewer, Kelly D. Goodwin, Christopher D. Sinigalliano, Annie M. Cox, David Wanless, Jakob Bartkowiak, Darcy L. Ebentier, Kaitlyn T. Hanley, Jared Ervin, Louise A. Deering, Orin C. Shanks, Lindsay A. Peed, Wim G. Meijer, John F. Griffith, Jorge SantoDomingo, Jennifer A. Jay, Patricia A. Holden, Stefan Wuertz^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

41 Citations (Scopus)

Abstract

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.

Original language	English
Pages (from-to)	6909-6920
Number of pages	12
Journal	Water Research
Volume	47
Issue number	18
DOIs	https://doi.org/10.1016/j.watres.2013.03.062
Publication status	Published - Nov 15 2013
Externally published	Yes

ASJC Scopus Subject Areas

Environmental Engineering
Civil and Structural Engineering
Ecological Modelling
Water Science and Technology
Waste Management and Disposal
Pollution

Keywords

Bacteroidales
Canine fecal pollution
Cp
Ct
DNQ
FIB
LLOQ
LOD
Microbial source tracking
MST
ND
PCR
QPCR
Quantitative PCR
ROQ
SIPP
TMDL

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10.1016/j.watres.2013.03.062

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Schriewer, A., Goodwin, K. D., Sinigalliano, C. D., Cox, A. M., Wanless, D., Bartkowiak, J., Ebentier, D. L., Hanley, K. T., Ervin, J., Deering, L. A., Shanks, O. C., Peed, L. A., Meijer, W. G., Griffith, J. F., SantoDomingo, J., Jay, J. A., Holden, P. A., & Wuertz, S. (2013). Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study. Water Research, 47(18), 6909-6920. https://doi.org/10.1016/j.watres.2013.03.062

@article{07a900bbec1d43bf89a524f232872676,

title = "Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study",

abstract = "The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.",

keywords = "Bacteroidales, Canine fecal pollution, Cp, Ct, DNQ, FIB, LLOQ, LOD, Microbial source tracking, MST, ND, PCR, QPCR, Quantitative PCR, ROQ, SIPP, TMDL",

author = "Alexander Schriewer and Goodwin, {Kelly D.} and Sinigalliano, {Christopher D.} and Cox, {Annie M.} and David Wanless and Jakob Bartkowiak and Ebentier, {Darcy L.} and Hanley, {Kaitlyn T.} and Jared Ervin and Deering, {Louise A.} and Shanks, {Orin C.} and Peed, {Lindsay A.} and Meijer, {Wim G.} and Griffith, {John F.} and Jorge SantoDomingo and Jay, {Jennifer A.} and Holden, {Patricia A.} and Stefan Wuertz",

year = "2013",

month = nov,

day = "15",

doi = "10.1016/j.watres.2013.03.062",

language = "English",

volume = "47",

pages = "6909--6920",

journal = "Water Research",

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Schriewer, A, Goodwin, KD, Sinigalliano, CD, Cox, AM, Wanless, D, Bartkowiak, J, Ebentier, DL, Hanley, KT, Ervin, J, Deering, LA, Shanks, OC, Peed, LA, Meijer, WG, Griffith, JF, SantoDomingo, J, Jay, JA, Holden, PA & Wuertz, S 2013, 'Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study', Water Research, vol. 47, no. 18, pp. 6909-6920. https://doi.org/10.1016/j.watres.2013.03.062

TY - JOUR

T1 - Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

AU - Schriewer, Alexander

AU - Goodwin, Kelly D.

AU - Sinigalliano, Christopher D.

AU - Cox, Annie M.

AU - Wanless, David

AU - Bartkowiak, Jakob

AU - Ebentier, Darcy L.

AU - Hanley, Kaitlyn T.

AU - Ervin, Jared

AU - Deering, Louise A.

AU - Shanks, Orin C.

AU - Peed, Lindsay A.

AU - Meijer, Wim G.

AU - Griffith, John F.

AU - SantoDomingo, Jorge

AU - Jay, Jennifer A.

AU - Holden, Patricia A.

AU - Wuertz, Stefan

PY - 2013/11/15

Y1 - 2013/11/15

N2 - The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.

AB - The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.

KW - Bacteroidales

KW - Canine fecal pollution

KW - Cp

KW - Ct

KW - DNQ

KW - FIB

KW - LLOQ

KW - LOD

KW - Microbial source tracking

KW - MST

KW - ND

KW - PCR

KW - QPCR

KW - Quantitative PCR

KW - ROQ

KW - SIPP

KW - TMDL

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UR - http://www.scopus.com/inward/citedby.url?scp=84887153986&partnerID=8YFLogxK

U2 - 10.1016/j.watres.2013.03.062

DO - 10.1016/j.watres.2013.03.062

M3 - Article

C2 - 23916711

AN - SCOPUS:84887153986

SN - 0043-1354

VL - 47

SP - 6909

EP - 6920

JO - Water Research

JF - Water Research

IS - 18

ER -

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

Abstract

ASJC Scopus Subject Areas

Keywords

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