2010
DOI: 10.1128/aem.02339-09
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PCR Inhibitor Levels in Concentrates of Biosolid Samples Predicted by a New Method Based on Excitation-Emission Matrix Spectroscopy

Abstract: Biosolids contain a wide variety of organic contaminants that are known for their ability to inhibit PCR. During sample processing, these contaminants are coconcentrated with microorganisms. Elevated concentrations of these compounds in concentrates render samples unsuitable for molecular applications. Glycine-based elution and recovery methods have been shown to generate samples with fewer PCR inhibitory compounds than the current U.S. EPA-recommended method for pathogen recovery from biosolids. Even with gly… Show more

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Cited by 52 publications
(37 citation statements)
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“…In contrast, by determining the global recovery rates for each sample from spiked parallel samples, a more realistic calculation of the actual V. cholerae abundance in the environment should be possible. In our experiments, special attention was paid to a variety of parameters responsible for potential influence on DNA recovery or PCR inhibition (40). Along a turbidity gradient ranging from drinking water to extremely turbid soda lake water, global recovery was most strongly impacted by the amount of DOC.…”
Section: Discussionmentioning
confidence: 99%
“…In contrast, by determining the global recovery rates for each sample from spiked parallel samples, a more realistic calculation of the actual V. cholerae abundance in the environment should be possible. In our experiments, special attention was paid to a variety of parameters responsible for potential influence on DNA recovery or PCR inhibition (40). Along a turbidity gradient ranging from drinking water to extremely turbid soda lake water, global recovery was most strongly impacted by the amount of DOC.…”
Section: Discussionmentioning
confidence: 99%
“…Therefore, characterization of inhibitors by analyzing raw-water sample and final sample concentrates is required for future establishment of a method to improve virus detection efficiency. Rock et al (26) applied EEM analysis to virus extracts obtained from biosolid samples using beef extract and glycine buffer and found peaks of humic acid-like and fulvic acidlike substances. However, the relationship between the virus detection efficiency and the EEM peaks was unclear.…”
Section: Discussionmentioning
confidence: 99%
“…However, RT-PCR inhibition has been reported to be observed even under these conditions (11,15), probably because of coconcentration of humic acid, which precipitates in the presence of cations (6) or acid conditions and becomes soluble at alkaline pH (23). Moreover, a silica membrane-based nucleic acid extraction/ purification method, which is a commonly used method for molecular detection of viruses in aquatic environments (3,15,41), cannot always effectively remove humic acid from samples (3,34). The principle of this method is based on attachment and detachment of nucleic acids to silica by altering pH and ionic strength (29).…”
mentioning
confidence: 99%
“…Such inhibitory effects also occur in the detection of bacterial genome (7,45). Humic acid, fulvic acid, a humic acid-like component in beef extract, which can be used as an eluent for virus concentration, and cations such as Ca 2ϩ and Fe 3ϩ are known to inhibit RT-PCR (1,15,26,34,49). Microfiltration (MF) membrane-based methods using acid rinse procedures followed by alkaline elution were developed to avoid the use of beef extract (16,21).…”
mentioning
confidence: 99%