Optimized DNA Extraction and Purification Method for Characterization of Bacterial and Fungal Communities in Lung Tissue Samples

Magne, F; Ruiz-Ruiz, Susana; Ponce, Carolina A.; Bustamante, R.; Martin, Viviana San; Gutierrez, Mireya; Gatti, Gianna; Vargas, Sergio L.; Moya, Andrés

doi:10.2139/ssrn.3496909

Cited by 3 publications

(4 citation statements)

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“…Currently, PCI is the most common and cost-efficient method for DNA isolation used in microbiome studies. The method claims to deliver high yielding and quality DNA [ 25 ]. Here we tested PCI isolation method to examine the latter claim and compare to an extraction kit to establish a standard protocol for DNA extraction of low microbial biomass samples.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, variability of normal lung microbiome of barrier laboratory mice includes not only the effects of strain, housing conditions and vendor [ 22 , 23 ], but full contribution of sample pre-treatment is poorly studied [ 24 ]. It has been suggested that Phenol Chloroform Isoamyl alcohol (PCI) method is suitable for the low-biomass lung tissue investigation [ 25 ], while others have pointed out it leads to artefactual results in low-biomass specimens and is the most labor-intensive [ 26 ]. These two reports published last year emphasize the lack of cohesion in the matter.…”

Section: Introductionmentioning

confidence: 99%

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An optimized approach for processing of frozen lung and lavage samples for microbiome studies

et al. 2022

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The respiratory tract has a resident microbiome with low biomass and limited diversity. This results in difficulties with sample preparation for sequencing due to uneven bacteria-to-host DNA ratio, especially for small tissue samples such as mouse lungs. We compared effectiveness of current procedures used for DNA extraction in microbiome studies. Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected to test different forms of sample pre-treatment and extraction methods to increase bacterial DNA yield and optimize library preparation. DNA extraction using a pre-treatment method of mechanical lysis (lung tissue) and one-step centrifugation (BALF) increased DNA yield and bacterial content of samples. In contrast, a significant increase of environmental contamination was detected after phenol chloroform isoamyl alcohol (PCI) extraction and nested PCR. While PCI has been a standard procedure used in microbiome studies, our data suggests that it is not efficient for DNA extraction of frozen low biomass samples. Finally, a DNA Enrichment kit was tested and found to improve the 16S copy number of lung tissue with a minor shift in microbial composition. Overall, we present a standardized method to provide high yielding DNA and improve sequencing coverage of low microbial biomass frozen samples with minimal contamination.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An optimized approach for processing of frozen lung and lavage samples for microbiome studies

et al. 2022

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“…The published literature has suggested that the complete lysis of fungal cell walls through beating can make a significant impact on yielded results. 28 Beating is a mechanical method to disrupt the cell wall that is performed prior to standard DNA extraction. In this step, ceramic or glass beads are added to the tube containing clinical samples.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of different DNA extraction methods based on steel‐bullet beating for molecular diagnosis of onychomycosis

Motamedi

Amini

Yazdanpanah

et al. 2022

Clinical Laboratory Analysis

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Background Considering increased trends toward molecular methods for detection/identification of fungi causing onychomycosis, the aim of this study is comparison three DNA extraction methods based on steel‐bullet beating to extract DNA from nail. Methods Ex ‐vivo onychomycosis model was developed using bovine hoof with Candida albicans and Aspergillus flavus. For two models, total DNA was extracted using the three different methods. In method 1, the extraction and purification were performed by steel‐bullet beating and phenol chloroform protocol, respectively. In method 2, a freezing step were applied before beating. The purification step in method 3 was carried out using a commercial kit, although DNA extraction was done similarly to method 1 in that approach. To evaluate the efficacy of each method, the extracted genomic DNA was amplified with Polymerase Chain Reaction (PCR) using Internal Transcribed Spacer (ITS) regions. Moreover, 50 nail samples were evaluated for onychomycosis using direct microscopy examination as well as PCR in order to evaluate the diagnostic efficiency of the optimal DNA extraction method. Results Regarding the desirable quality of the extracted DNA, cost effectiveness, and simplicity, method 1 could be used to extract DNA effectively. Additionally, the obtained data showed that PCR had a higher detection rate of fungal agents in the nail samples than direct microscopic examination. Conclusions This study demonstrated that the mechanical disruption of the cell wall by steel‐bullet beating is a useful and practical method to improve the quantity and quality of fungal DNA thorough the extraction process.

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“…Even though several microorganisms associated with numerous marine organisms have been broadly studied, the microbial communities associated with nudibranchs are relatively unknown. As molecular biology techniques are developing into more valuable tools for understanding microbial communities and their structure from an ecological perspective, it is imperative to produce pure DNA and overcome the tissue problem that challenges the DNA extraction process [2][3][4][5][6]. In contrast, pure DNA is a base for further molecular genetic analyses [7,8], like PCR and real-time PCR analysis, next-generation sequencing, cloning, and other genotyping procedures.…”

Section: Introductionmentioning

confidence: 99%

Detection of Nudibranch Chromodoris quadricolor associated microbiome using two DNA extraction methods

Abdelrahman

Hanora

Aboseidah

et al. 2021

Frontiers in Scientific Research and Technology

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DNA extraction with high quality is critical to all molecular genetic analyses. However, obtaining DNA from microbes associated with animals is challenging. Despite the availability of various DNA extraction kits in the markets, no studies were conducted to date to evaluate their potential for the invertebrates such as nudibranch, one of Mollusca. This study compared the Quick-DNA Fungal/Bacterial Miniprep Kit and CTAB methods and tested them across four samples of Chromodoris quadricolor gut and skin tissues. The universal bacterial primers 331f and 797r and the animalspecific primers LCO1490-JJ and HCO2198-JJ were used to amplify the 16S rRNA gene and partial mitochondrial cytochrome oxidase I gene using extracted DNA as a template. The DNA and PCR products' quality and concentration were verified with agarose gel and Nanodrop, respectively. The two methods' quality assessed using the deep pyrosequencing of the 16S rRNA gene in capturing a more diverse microbiome. The highest yield and purity (over 1000 ng / µL) were obtained with the CTAB method, while it was not exceeded 260 ng/µL with Quick DNA kit and display high purity. Also, 16S rRNA community amplicon sequencing revealed that the CTAB way could catch more diverse bacterial groups. The most efficient method of DNA extraction was CTAB, as it achieved both high concentration and purity.

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Optimized DNA Extraction and Purification Method for Characterization of Bacterial and Fungal Communities in Lung Tissue Samples

Cited by 3 publications

References 0 publications

An optimized approach for processing of frozen lung and lavage samples for microbiome studies

An optimized approach for processing of frozen lung and lavage samples for microbiome studies

Evaluation of different DNA extraction methods based on steel‐bullet beating for molecular diagnosis of onychomycosis

Detection of Nudibranch Chromodoris quadricolor associated microbiome using two DNA extraction methods

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