The NSF Public Access Repository (PAR) system and access will be unavailable from 11:00 PM ET on Thursday, February 13 until 2:00 AM ET on Friday, February 14 due to maintenance. We apologize for the inconvenience.
Explore Research Products in the PAR It may take a few hours for recently added research products to appear in PAR search results.
Title: An assessment of three methods for extracting bacterial DNA from beach sand
AbstractAims
Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method.
Methods and results
The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20–80 gene copies g−1, recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement.
Conclusions
Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis.
Significance and impact of study
Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches.
Acheamfour, Chanelle L; Parveen, Salina; Gutierrez, Alan; Handy, Eric T; Behal, Sara; Kim, Donghyun; Kim, Seongyun; East, Cheryl; Xiong, Ray; Haymaker, Joseph R; et al(
, Microbiology Spectrum)
Wang, Luxin
(Ed.)
ABSTRACT
Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens likeSalmonella entericaandListeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detectionof S. enterica and L. monocytogenesby qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n= 1,990) was analyzed by multiplex qPCR specific forS. entericaandL. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection ofS. entericaandL. monocytogeneswere 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter forS. enterica; forL. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for bothS. entericaandL. monocytogenes, indicating that water type may influence the agreement of these results.
IMPORTANCE
Detecting foodborne pathogens in irrigation water can inform interventions and management strategies to reduce risk of contamination and illness associated with fresh and fresh-cut fruits and vegetables. The use of non-culture methods like qPCR has the potential to accelerate the testing process. Results indicated that pond and reclaimed water showed higher levels of agreement between culture and qPCR methods than river water, perhaps due to specific physiochemical characteristics of the water. These findings also show that season and sample volume affect the agreement of qPCR and culture results. Overall, qPCR methods could be more confidently utilized to determine the absence ofSalmonella entericaandListeria monocytogenesin irrigation water samples examined in this study.
There is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications.
Results
In a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant,Arabidopsis thaliana(L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastidrbcLand nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis.
Conclusions
The method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.
Johnson, Gabriel; Canty, Steven W.; Lichter‐Marck, Isaac H.; Wagner, Warren; Wen, Jun(
, Applications in Plant Sciences)
AbstractPremise
The preservation of plant tissues in ethanol is conventionally viewed as problematic. Here, we show that leaf preservation in ethanol combined with proteinase digestion can provide high‐quality DNA extracts. Additionally, as a pretreatment, ethanol can facilitate DNA extraction for recalcitrant samples.
Methods
DNA was isolated from leaves preserved with 96% ethanol or from silica‐desiccated leaf samples and herbarium fragments that were pretreated with ethanol. DNA was extracted from herbarium tissues using a special ethanol pretreatment protocol, and these extracts were compared with those obtained using the standard cetyltrimethylammonium bromide (CTAB) method.
Results
DNA extracted from tissue preserved in, or pretreated with, ethanol was less fragmented than DNA from tissues without pretreatment. Adding proteinase digestion to the lysis step increased the amount of DNA obtained from the ethanol‐pretreated tissues. The combination of the ethanol pretreatment with liquid nitrogen freezing and a sorbitol wash prior to cell lysis greatly improved the quality and yield of DNA from the herbarium tissue samples.
Discussion
This study critically reevaluates the consequences of ethanol for plant tissue preservation and expands the utility of pretreatment methods for molecular and phylogenomic studies.
SARS-CoV-2 is an RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Viruses exist in complex microbial environments, and recent studies have revealed both synergistic and antagonistic effects of specific bacterial taxa on viral prevalence and infectivity. We set out to test whether specific bacterial communities predict SARS-CoV-2 occurrence in a hospital setting.
Methods
We collected 972 samples from hospitalized patients with COVID-19, their health care providers, and hospital surfaces before, during, and after admission. We screened for SARS-CoV-2 using RT-qPCR, characterized microbial communities using 16S rRNA gene amplicon sequencing, and used these bacterial profiles to classify SARS-CoV-2 RNA detection with a random forest model.
Results
Sixteen percent of surfaces from COVID-19 patient rooms had detectable SARS-CoV-2 RNA, although infectivity was not assessed. The highest prevalence was in floor samples next to patient beds (39%) and directly outside their rooms (29%). Although bed rail samples more closely resembled the patient microbiome compared to floor samples, SARS-CoV-2 RNA was detected less often in bed rail samples (11%). SARS-CoV-2 positive samples had higher bacterial phylogenetic diversity in both human and surface samples and higher biomass in floor samples. 16S microbial community profiles enabled high classifier accuracy for SARS-CoV-2 status in not only nares, but also forehead, stool, and floor samples. Across these distinct microbial profiles, a single amplicon sequence variant from the genusRothiastrongly predicted SARS-CoV-2 presence across sample types, with greater prevalence in positive surface and human samples, even when compared to samples from patients in other intensive care units prior to the COVID-19 pandemic.
Conclusions
These results contextualize the vast diversity of microbial niches where SARS-CoV-2 RNA is detected and identify specific bacterial taxa that associate with the viral RNA prevalence both in the host and hospital environment.
Wenger, Kelsey; Pendleton, Chandler; Xie, Xian Jin; Caplan, Daniel J.; Drake, David; Marchini, Leonardo(
, Special Care in Dentistry)
AbstractPurpose/aim
To analyze potential factors associated with levels of selected oral pathogens, as well as total aerobic bacterial species, among nursing home residents.
Materials and methods
Nursing home residents were divided into three groups (G1 included people with teeth but no dentures, G2 included people with teeth and dentures, and G3 included people with no teeth and with dentures). All participants had microbiological samples collected from their oral cavity and dentures. Counts of total aerobic bacterial species,Porphyromonas gingivalis,Fusobacterium nucleatum,Actinomyces viscosus,Aggregatibacter actinomycetemcomitans, andCandida albicanswere compared among groups using the Wilcoxon rank sum test. A multivariate analysis was also performed to control other available covariates.
Results
Bivariate analysis revealed significant differences among the groups, and multivariate analysis showed that sex, the presence of natural teeth, denture wearing, oral hygiene indices, and systemic health conditions were associated with bacterial andCandida albicanslog counts.
Conclusions
Presence of natural teeth and denture wearing, as well as oral hygiene, sex and systemic health conditions were associated with bacterial andCandida albicanslog counts among nursing home residents.
Gallard-Gongora, Javier, Lobos, Aldo, Conrad, James W., Peraud, Jayme, and Harwood, Valerie J. An assessment of three methods for extracting bacterial DNA from beach sand. Journal of Applied Microbiology 132.4 Web. doi:10.1111/jam.15423.
Gallard-Gongora, Javier, Lobos, Aldo, Conrad, James W., Peraud, Jayme, & Harwood, Valerie J. An assessment of three methods for extracting bacterial DNA from beach sand. Journal of Applied Microbiology, 132 (4). https://doi.org/10.1111/jam.15423
Gallard-Gongora, Javier, Lobos, Aldo, Conrad, James W., Peraud, Jayme, and Harwood, Valerie J.
"An assessment of three methods for extracting bacterial DNA from beach sand". Journal of Applied Microbiology 132 (4). Country unknown/Code not available: Oxford University Press. https://doi.org/10.1111/jam.15423.https://par.nsf.gov/biblio/10391749.
@article{osti_10391749,
place = {Country unknown/Code not available},
title = {An assessment of three methods for extracting bacterial DNA from beach sand},
url = {https://par.nsf.gov/biblio/10391749},
DOI = {10.1111/jam.15423},
abstractNote = {Abstract AimsBeach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. Methods and resultsThe analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20–80 gene copies g−1, recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. ConclusionsAmong the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. Significance and impact of studyStandardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches.},
journal = {Journal of Applied Microbiology},
volume = {132},
number = {4},
publisher = {Oxford University Press},
author = {Gallard-Gongora, Javier and Lobos, Aldo and Conrad, James W. and Peraud, Jayme and Harwood, Valerie J.},
}
Warning: Leaving National Science Foundation Website
You are now leaving the National Science Foundation website to go to a non-government website.
Website:
NSF takes no responsibility for and exercises no control over the views expressed or the accuracy of
the information contained on this site. Also be aware that NSF's privacy policy does not apply to this site.