Accurate qPCR quantification in polymicrobial communities requires assessment of gDNA extraction efficiency

Cerca, Nuno; Lima, Ângela; França, Ângela

doi:10.1016/j.mimet.2022.106421

Cited by 6 publications

(7 citation statements)

References 17 publications

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“…3a). This was previously demonstrated by Cerca and colleagues (Cerca et al, 2022) Several previous ecology studies noted the potential influence of DNA extraction methods on DNA yield, bacterial diversity and relative abundance measurements, for example in soil samples (Martin-Laurent et al, 2001), human breast milk (Douglas et al, 2020) and faecal samples (Hart et al, 2015).…”

Section: The Choice Of the Dna Extraction Methods Is Importantsupporting

confidence: 53%

The impact of DNA extraction on the quantification ofLegionella, with implications for ecological studies

Cavallaro,

Gabrielli,

Hammes

et al. 2024

Preprint

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Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques such as DNA extraction differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which was reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterise the associated microbial community.

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Section: The Choice Of the Dna Extraction Methods Is Importantsupporting

confidence: 53%

The impact of DNA extraction on the quantification ofLegionella, with implications for ecological studies

Cavallaro,

Gabrielli,

Hammes

et al. 2024

Preprint

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“…Without normalization of the different gDNA extraction efficiencies, performing calibration curves for qPCR quantification would result in significant quantification errors, as we have previously shown (Cerca et al, 2022). Not surprisingly, gDNA extraction with the phenol/chloroform method yielded more gDNA than the silica-column based approach (Ibrahim and Nassar, 2018;Schiebelhut et al, 2017), except at very low bacterial concentrations, where both extraction methods yielded similar results.…”

Section: J O U R N a L P R E -P R O O Fmentioning

confidence: 79%

“…We previously observed that, for lower bacterial concentrations, a luciferase cDNA exogenous control was not sufficient to accurately quantify bacterial populations in three species consortia, when using a silica-column based extraction commercial kit (Cerca et al, 2022). We hypothesized that the low DNA mass of such small exogenous controls could result in a very low recovery rate in the DNA optimized silica columns commonly used for genomic DNA (gDNA) extraction, which could bias quantification.…”

mentioning

confidence: 99%

“…Bacterial suspensions with culture densities ranging from 3 × 10 8 cells.mL-1 to 1 × 10 7 cells.mL-1 were prepared in PBS 1x. Each culture was divided in equal aliquots and gDNA was extracted with either the DNeasy Ultraclean Microbial Kit (QIAGEN), using the manufacturer's instructions with minor modifications (Cerca et al, 2022), or with the classical phenol/chloroform method (Kumar and Mugunthan, 2018). A mixture containing three different exogenous controls, namely, commercially available luciferase cDNA (Promega) (10 µL of 10 8 copies of luciferase cDNA, with 67 bp), 10 µL of 1 ng.µL-1 Staphylococcus epidermidis gDNA (2.6 Mb, (NCBI, 2005), extracted with DNeasy Ultraclean), and 10 µL of 1 ng.µL-1 of a plasmid (piMAY, 5473 bp (Monk et al, 2012)), was spiked before DNA isolation to determine the % of DNA recovered, in both methods.…”

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confidence: 99%

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Assessing recovery rates of distinct exogenous controls for gDNA extraction efficiency using phenol-chloroform or silica-column based extractions

Lima

Sousa

Macedo

et al. 2022

Journal of Microbiological Methods

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“…Genomic DNA (gDNA) from the in vitro and ex vivo polymicrobial biofilms as well as from pure cultures of the six species (for the qPCR calibration curves), were performed as described before ( Cerca et al., 2022 ). To assess the efficiency and variability of gDNA extraction between samples, 10 µL of luciferase complementary DNA (cDNA), obtained as described before ( Magalhães et al., 2019 ), was added to each sample before proceeding with gDNA isolation.…”

Section: Methodsmentioning

confidence: 99%

Six Bacterial Vaginosis-Associated Species Can Form an In Vitro and Ex Vivo Polymicrobial Biofilm That Is Susceptible to Thymbra capitata Essential Oil

Roșca

Castro

Sousa

et al. 2022

Front. Cell. Infect. Microbiol.

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Bacterial vaginosis (BV) is associated with serious gynaecologic and obstetric complications. The hallmark of BV is the presence of a polymicrobial biofilm on the vaginal epithelium, but BV aetiology is still a matter of debate. We have previously developed an in vitro biofilm model that included three BV-associated species, but, up to now, no studies are available whereby more bacterial species are grown together to better mimic the in vivo situation. Herein, we characterized the first polymicrobial BV biofilm consisting of six cultivable BV-associated species by using both in vitro and ex vivo vaginal tissue models. Both models revealed that the six species were able to incorporate the polymicrobial biofilm, at different bacterial concentrations. As it has been thought that this polymicrobial biofilm may increase the survival of BV-associated species when exposed to antibiotics, we also assessed if the Thymbra capitata essential oil (EO), which has recently been shown to be highly bactericidal against several Gardnerella species, could maintain its anti-biofilm activity against this polymicrobial biofilm. Under our experimental conditions, T. capitata EO exhibited a high antibacterial effect against polymicrobial biofilms, in both tested models, with a significant reduction in the biofilm biomass and the number of culturable cells. Overall, this study shows that six BV-associated species can grow together and form a biofilm both in vitro and when using an ex vivo model. Moreover, the data obtained herein should be considered in further applications of T. capitata EO as an antimicrobial agent fighting BV.

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Accurate qPCR quantification in polymicrobial communities requires assessment of gDNA extraction efficiency

Cited by 6 publications

References 17 publications

The impact of DNA extraction on the quantification ofLegionella, with implications for ecological studies

The impact of DNA extraction on the quantification ofLegionella, with implications for ecological studies

Assessing recovery rates of distinct exogenous controls for gDNA extraction efficiency using phenol-chloroform or silica-column based extractions

Six Bacterial Vaginosis-Associated Species Can Form an In Vitro and Ex Vivo Polymicrobial Biofilm That Is Susceptible to Thymbra capitata Essential Oil

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