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Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

Overview of attention for article published in Water Research, June 2016
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Title
Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR
Published in
Water Research, June 2016
DOI 10.1016/j.watres.2016.03.026
Pubmed ID
Authors

Joel P. Stokdyk, Aaron D. Firnstahl, Susan K. Spencer, Tucker R. Burch, Mark A. Borchardt

Abstract

The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay alone rather than the entire sample process. Our objective was to develop an approach to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) into each procedural step working backwards (i.e., extraction, secondary concentration, primary concentration), which established a concentration that was detectable following losses of the pathogen from processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then third, concentration. If the fraction of positive replicates equaled 1 or 0 for two concentrations, we selected another. We calculated the LOD using probit analysis. To demonstrate our approach we determined the 95% LOD for Salmonella enterica serovar Typhimurium, adenovirus 41, and vaccine-derived poliovirus Sabin 3, which were 11, 12, and 6 genomic copies (gc) per reaction (rxn), respectively (equivalent to 1.3, 1.5, and 4.0 gc L(-1) assuming the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple genetic targets simultaneously (i.e., spiking a single sample with multiple microorganisms). An LOD determined this way can facilitate study design, guide the number of required technical replicates, aid method evaluation, and inform data interpretation.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 57 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
United States 2 4%
Slovenia 1 2%
Unknown 54 95%

Demographic breakdown

Readers by professional status Count As %
Researcher 11 19%
Student > Ph. D. Student 11 19%
Student > Master 9 16%
Other 6 11%
Student > Bachelor 3 5%
Other 9 16%
Unknown 8 14%
Readers by discipline Count As %
Environmental Science 10 18%
Engineering 9 16%
Agricultural and Biological Sciences 9 16%
Biochemistry, Genetics and Molecular Biology 6 11%
Immunology and Microbiology 3 5%
Other 6 11%
Unknown 14 25%

Attention Score in Context

This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 28 March 2016.
All research outputs
#7,366,667
of 12,269,818 outputs
Outputs from Water Research
#3,518
of 5,590 outputs
Outputs of similar age
#143,818
of 282,068 outputs
Outputs of similar age from Water Research
#55
of 200 outputs
Altmetric has tracked 12,269,818 research outputs across all sources so far. This one is in the 37th percentile – i.e., 37% of other outputs scored the same or lower than it.
So far Altmetric has tracked 5,590 research outputs from this source. They receive a mean Attention Score of 3.6. This one is in the 34th percentile – i.e., 34% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 282,068 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 45th percentile – i.e., 45% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 200 others from the same source and published within six weeks on either side of this one. This one has gotten more attention than average, scoring higher than 64% of its contemporaries.