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Article

A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses

by
Abdulkarim Shwani
1,
Bin Zuo
2,
Adnan Alrubaye
1,3,
Jiangchao Zhao
1,2 and
Douglas D. Rhoads
1,4,*
1
Program in Cell and Molecular Biology, University of Arkansas, Fayetteville, AR 72701, USA
2
Department of Animal Sciences, University of Arkansas, Fayetteville, AR 72701, USA
3
Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA
4
Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(1), 141; https://doi.org/10.3390/app14010141
Submission received: 29 November 2023 / Revised: 14 December 2023 / Accepted: 20 December 2023 / Published: 22 December 2023
(This article belongs to the Special Issue Applied Microbial Biotechnology for Poultry Science)
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Abstract

:
DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract total bacterial DNA from environmental samples based on the sodium hydroxide lysis of cells with or without capture by magnetic beads for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in as little as 10 min at room temperature with dilution, or the DNA can be further purified using silica-coated paramagnetic beads. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrated that an impinger and portable pump can efficiently capture bacteria from the air in poultry facilities for rapid NaOH extraction to quantify total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well suited for routine, high-throughput screening and for metagenomic analyses for specific pathogens, even in resource-limited situations.

1. Introduction

Reliable and accurate molecular diagnostics are crucial for the detection of pathogenic bacteria in clinical, food, and environmental samples [1,2,3]. There are various methods and commercial kits available for the rapid extraction of bacterial DNA from environmental samples for polymerase chain reaction (PCR) [4,5,6,7]. These methods achieve lysis by heat treatment, detergent, enzymes, pulverization, pH, chaotropic chemicals, or a combination of these, which is then followed by either chaotrope-based fractionation, phenol extraction, or size exclusion column purification [8,9,10,11,12]. Purification of the extracted DNA can involve precipitation or column-based nucleic acid capture. Traditionally, DNA extraction from cultured bacteria often includes expensive and complicated enzyme combinations or costly hazardous chemicals such as phenol or chloroform [12,13,14,15]. These methods vary in effectiveness depending on the application and the matrix from which the cells are obtained [16]. Although current techniques are well proven to produce high-quality nucleic acids (DNA or RNA), they are time-consuming, labor-intensive, and expensive to implement. In addition, the quantity of genomic DNA that they produce could be insufficient. These limitations become much more significant in settings with limited resources. As a result, the implementation of molecular testing is considerably hampered in many regions of the globe, especially in developing countries [12]. Therefore, the development of rapid, easy-to-use, and more efficient DNA extraction techniques that do not rely on complex laboratory equipment facilitates the advancement of molecular clinical detection technologies.
Recent years have seen the development of novel approaches for extracting DNA from plants [17], mouse tails [18], microbes from sediments [19,20], protozoa [21], and fungal/oomycete samples [22] using NaOH alone or in combination with other chemicals. Therefore, it has been proven that NaOH is capable of effectively lysing a variety of cell types from diverse biological materials within 10 min. The extraction is most effective using NaOH, which also inactivates nucleases during the extraction process. In most of these treatments, NaOH was administered along with reagents including sodium dodecyl sulfate or sodium acetate [17,23] or at elevated temperatures to disrupt cell walls, especially for Gram-positive bacteria [20,24]. Hence, these treatments complicate the protocol with additional PCR inhibitors or other time-consuming steps. Consequently, a fast DNA extraction procedure that consistently produces high DNA yields and can be applied to both Gram-positive and Gram-negative bacteria without the use of additional chemicals or enzymatic treatments would be beneficial in terms of saving time and money while allowing for increased DNA yields.
The goal of this research was to develop an inexpensive technique for rapid lysis of bacteria that can be carried out with minimal laboratory equipment and used for subsequent DNA-based diagnostics. Our impetus was monitoring for specific bacteria in the environment during our lameness trials. In these trials, we showed that bacterial chondronecrosis with osteomyelitis (BCO) lameness can spread through the air from one pen to others [25,26]. We previously used open agar plates for culture-based sampling of air during lameness trials [25]. However, culture-based sampling is less efficient than qPCR analysis with genus- or species-specific primers. We found that we could use a standard glass impinger to sample air for viable bacterial plate counts in our lameness trial facilities. However, extracting DNAs from these same samples was more problematic, especially for some species of concern in our experiments. We found that simple alkaline extraction can be effective for liberation of DNA from many Gram-positive and -negative bacteria. Either the alkaline extract can be diluted sufficiently for direct qPCR or the DNA can be directly captured from the alkaline solution using paramagnetic beads. We found that the NaOH method was effective for bacterial DNA extraction from a variety of different environmental samples, including air, soil, sewage water, food, environmental surfaces, chicken cloacal swabs, broth cultures, plate colonies, and swine fecal samples. The optimized DNA preparation technique could be a substitute for the current expensive protocols and commercial DNA extraction kits for the detection of various bacterial targets.

2. Materials and Methods

2.1. Reagents and Materials

Purified water was >18 MΩ from a Barnstead™ GenPure™ Pro Water Purification System (Thermo-Fisher Scientific, Waltham, MA, USA). Sterile pure water (SPW) was autoclaved purified water. Stocks of 1 M NaOH were prepared in SPW from solid (S318-3, Thermo-Fisher Scientific). Sterile swabs were autoclaved cotton-tip swabs (Puritan LLC, Guilford, ME, USA). Rectal samples were collected with an Opti-Swab® Liquid Amies Collection & Transport System (Puritan LLC). Paramagnetic beads were Mag-Bind RXN Pure Plus (Omega Bio-Tek, Norcross, GA, USA) and PureSil-Silica Magnetic Beads (BioChain, Newark, CA, USA). The Mag-Bind RXN Pure Plus are provided in binding buffer. For the PureSil-Silica beads, the bead-binding buffer (BBB) was 80% isopropanol, 800 mM guanidine isothiocyanate, 8 mM TrisCl pH 8, 3.52 mM Na2EDTA (BioChain). The commercial kit for DNA extraction from rectal fecal swabs was a PowerLyzer PowerSoil DNA Isolation Kit (Qiagen, Hilden, Germany).

2.2. Bacterial Cultures and Growth

Bacterial stocks were stored at −80 °C in 40% glycerol. Media included CHROMagar Orientation (CO) and CHROMagar Staphylococcus (CS) (DRG International, Inc., Springfield, NJ, USA) and tryptic soy broth (TSB) and nutrient broth (NB) (Difco Laboratories, Franklin Lakes, NJ, USA). Difco bacteriological agar was added at 1.5% for solidification when necessary. Overnight cultures were diluted 1:100 in broth. Absorbance at 650 nm was used to compute the cell density based on a precalibrated formula:
CFU/mL = (A650 × 109) + 106

2.3. Alkaline Extraction from Environmental or Bacterial Culture Sample Preparation

For air samples: The air was collected by bubbling through 20 mL of 0.9% saline in a 30 mL autoclave-sterilized glass impinger with sintered glass inlet tube (Chemglass Life Sciences LLC, Vineland, NJ, USA) using a portable pipet pump for 20 min. Liquid samples were then centrifuged at 4400× g for 10 min at 4 °C in a swinging bucket rotor. Pellets were resuspended by vortex in 90 µL of SPW then mixed with 10 µL 1 M NaOH. See below for extraction processing.
For soil samples: The top dried layer of the ground was removed to a depth of 2–5 cm using a sterile spatula. The underlying moist silt soil was collected in a sterile 50 mL conical tube. From the soil sample, 50 mg was transferred into a 1.5 mL tube containing 450 µL of SPW and vortexed for 30 s, followed by the addition of 50 µL of 1 M NaOH; then it was mixed and incubated for 10 min at RT. The sample was centrifuged at 8000× g for 5 min, and supernate was collected for extraction processing (see below).
For cloacal or fecal swab samples: Cloacae of day-old chicks were swabbed using autoclaved cotton swabs. Pig rectal swabs were swirled in 450 µL of SPW in a 1.5 mL tube and mixed with 50 µL of 1 M NaOH. See below for extraction processing.
For food samples: (a) The surface of a cheese block was swabbed using sterile cotton swabs then swirled in 450 µL of SPW in a 1.5 mL tube, and (b) 10 mg of bread was added to 450 µL of SPW in 1.5 mL tubes. In both cases, the tubes were vortexed for 30 s and mixed with 50 µL of 1 M NaOH. See below for extraction processing.
For environmental surfaces: Sterile cotton swabs were rubbed on surfaces then swirled in 450 µL of SPW in a 1.5 mL tube, which was then vortexed for 30 s and mixed with 50 µL of 1 M NaOH. See below for extraction processing.
For environmental liquid sampling: A 1 mL sample in a 1.5 mL tube was centrifuged at 8000× g for 10 min at 4 °C, and the was pellet resuspended in 450 µL of SPW and mixed with 50 µL of 1 M NaOH. See below for extraction processing.
Bacterial culture sampling: An amount of 10 µL from either a broth overnight culture or a single colony suspended in 25 µL of SPW from an overnight agar plate was mixed with 90 µL of 100 mM NaOH. See below for extraction processing.

2.4. Extraction Processing

The NaOH extraction was allowed to proceed for 10 min at RT and was then either stored at −20 °C, directly diluted with 4 volumes of Te (10 mM TrisCl pH 7.5, 0.1 mM EDTA), or processed for paramagnetic bead capture.
For Mag-Bind RXN Pure Plus beads, 100 µL of NaOH extract was mixed with 100 µL of the bead suspension. For PureSil beads, 100 µL of NaOH extract was mixed with 5 µL of resuspended beads and 95 µL of BBB. The extract–bead solution was mixed well and incubated at RT for 10 min. The beads were captured on a magnetic stand, and the solution was collected with a micropipettor and discarded. The beads were rinsed twice by addition of 200 µL RT 70% ethanol, and the beads were resuspended with a brief vortex, then magnetic capture was performed and the solution was discarded, being certain to remove all solution after the second rinse. The open tubes were allowed to air-dry for 5–10 min. The beads were suspended in 50 µL of Te and incubated for 5 min at RT, then magnetic capture was performed and the eluate transferred to a clean tube. Eluate was either directly subjected to qPCR or stored frozen at −20 °C.
For pig rectal swabs extracted with a commercial kit, a total of 100 µL swab solution was used for the extraction with a PowerLyzer PowerSoil DNA Isolation Kit (Qiagen, Hilden, Germany) according to the protocol. The extracted DNA was quantified with a NanoDrop (Thermo Fisher Scientific, Wilmington, DE, USA).

2.5. Quantification of Extracted DNA by qPCR-HRM

The diluted NaOH extracts or paramagnetic-bead-purified DNAs were quantified for total bacterial DNA using qPCR for 16S rDNA using primers 8F and 936R [27]. Reactions were run in triplicates of 20 µL in 1× Taq Buffer (50 mM Tris pH 8.3, 1.25 mM MgCl2, 300 ng/µL BSA), 0.2 mM dNTPs, 0.25 µM primers, 1× EvaGreen® Dye (Biotium, Fremont, CA, USA), 2 µL of extracted DNA, and 4 U Taq Polymerase in 96-well plates in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cycle parameters were as follows: 90 °C for 45 s, 5 cycles of 90 °C for 15 s, 71.5 °C for 15 s, and 72 °C for 60 s, followed by 35 cycles with the same parameters with a plate read after each cycle. The products were then subjected to high-resolution melt (HRM) analysis (to verify that any amplification signal was not artifactual) consisting of 72° C for 180 s, 90 °C for 60 s, 70 °C for 120 s, followed by a 70 °C to 90 °C melt with steps of 0.1 °C for 5 s and a plate read. The amplification and melt curves were analyzed using CFX Manager v 3.1 (Bio-Rad Laboratories).

2.6. Microbiome Sequencing of Extracted DNAs

Extracted DNAs from swine fecal samples were processed for microbiome analysis as described [28]. Briefly, the DNAs were amplified for 16S subsequences and submitted for barcoding and sequencing. Sequence reads were analyzed using Qiime2-2021.11, denoised by Deblur, and annotated with the Silva (version 138) database.

3. Results

3.1. Air Sampling during Bacterial Infection Lameness Trial

We previously found that waving open agar plates for several minutes could capture 50–300 CFU during our experiments [25,29]. The primary airborne bacteria during these experiments were typically Staphylococcus saprophyticus [25], while similar sampling on a commercial broiler farm were primarily Staphylococcus cohnii [29]. In order to improve the sampling and provide better quantitative data, we evaluated the use of a sterile impinger containing 20 mL of 0.9% saline and a battery-operated pipet pump as a portable air sampler for collecting viable bacteria during our BCO lameness trials. With the portable pump and impinger, we found that total CFU increased with the length of time sampled on a given day and generally increased with the day of the experiment, implying that the bacterial load in the air increased as the birds matured and the bacterial infection spread (Table 1). As we were interested in using qPCR to determine the prevalence of specific bacterial genera/species, we tried several standard methods for rapid liberation of PCR-ready DNA. These included boiling [30,31], sonication [32], and glass-bead beating [33]. The qPCR results were either negative or variable even though we had viable bacteria present in the sample (Table 1). Therefore, we evaluated other methods for liberating PCR-ready DNA from bacterial broth and plate cultures.

3.2. Optimization of DNA Extraction

Broth cultures and bacterial colonies for several different genera and species from our collection were tested for DNA liberation using (a) boiling, (b) sonication, (c) glass-bead beating, (d) enzymatic extraction [34], (e) lipid extraction with 1-ethyl-3-methylimidazolium acetate (C2minOAc) [12], and (f) NaOH lysis with sodium acetate neutralization [35]. Bacteria species tested included Enterococcus faecalis (three isolates), Enterococcus cecorum, Escherichia coli (two isolates), Escherchia fergusonii, Staphylococcus agnetis, Staphylococcus aureus (two isolates), Staphylococcus gallinarum, Staphylococcus nepalensis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus lentus, Staphylococcus equorum, Staphylococcus cohnii, Streptococcus agalactiae, and Salmonella enterica. Of the six methods tested, the boiling approach was the most reliable, as methods (b)–(f) were largely ineffective for many of the Gram-positive organisms. Particularly problematic for the boil method were the Enterococcus isolates, of which from multiple boil extractions only a few gave positive PCR for 16S rDNA amplification.
NaOH lysis is a common method for bacterial lysis, especially for plasmid isolation [13], and procedures have been published for extraction of plant, human, bacterial, and yeast DNA for PCR using NaOH and heat [17,19,24,36], so we investigated how to best process bacterial NaOH extracts for subsequent PCR. We first examined what the threshold concentration of NaOH is for inhibition of a standard qPCR reaction for 16S rDNA using purified S. agnetis DNA as the target. The results with our PCR buffer formulation (Methods) showed no inhibition at NaOH concentrations of 0 to 5.6 mM NaOH. The average Ct values increased by about one cycle with 7.5 mM NaOH and by approximately four with 10 mM NaOH. We then tested 25, 50, 75, and 100 mM NaOH in duplicate for 10 min room temperature (RT) extraction of 4 × 103 CFU of broth-grown S. agnetis 908 (see Methods). After incubation, each sample was diluted with four volumes of SPW, and qPCR-HRM was performed in triplicate on 2 µL of the diluted sample (final NaOH concentrations in the PCR were therefore 0.5, 1, 1.5, and 2 mM). The qPCR results in Figure 1 demonstrate that all four concentrations of NaOH liberated DNA but that 75 and 100 mM yielded lower Ct values, consistent with more complete lysis. We chose 100 mM as an appropriately effective concentration for subsequent extractions where fivefold dilution would reduce the concentration to 20 mM NaOH, with the subsequent addition of 2 µL to a 20 µL PCR, which would result in a final concentration of 2 mM NaOH in the PCR or less than half the minimum inhibitory concentration. We compared RT extraction to extraction at 37 °C and extraction for 5, 10, 20, or 30 min and found no difference for time or temperature. We added water-washed S. agnetis 908 from broth overnight cultures in 10-fold dilutions estimated at 8 × 10−2 to 8 × 105 CFU in 2 µL directly to 20 µL qPCR. Specific amplification of the 16S amplicon resulted from the reactions from 8 × 102 to 8 × 105, but the Ct values were higher for ≥8 × 104 CFU, consistent with PCR inhibition. When we instead used a similar dilution series for extraction with 100 mM NaOH followed by fivefold dilution, there was specific amplification with input of as little as 10 CFU and no inhibition even with input of estimated 107 CFU. Thus, NaOH extraction produced a specific qPCR-HRM product from fewer input cells and minimized PCR inhibition from the addition of more than one hundred times more cells.
We then compared the boil and NaOH extraction methods for an E. cecorum isolate (1415) and three different isolates of E. faecalis (1558, 1570, and 1582), which had previously been problematic for boil extractions for PCR. We included S. agnetis 908 as a control. Equivalent volumes of cell extracts from each method were added to the qPCR-HRM. The results in Figure 2 show that the NaOH extraction produced Ct values lower than those from the boil. For E. faecalis 1570, there was no 16S amplification from the boil extract, while the NaOH with dilution method was positive for 16S PCR. Based on ΔCt calculations [37] using the positive control of purified S. agnetis 908 genomic DNA, we computed the E. faecalis 1570 NaOH extract from 20 µL of overnight culture and dilution to a final volume of 500 µL to be 3 pg/µL. Similar calculations for the NaOH extracts for the other cultures were as follows: S. agnetis 908: 680 pg/µL; E. cecorum 1415: 71 pg/µL; E. faecalis 1558: 16 pg/µL; and E. faecalis 1572: 5 pg/µL. The NaOH yield for E cecorum 1415 was more than 30× higher than that of the boil method.
We speculated that the NaOH extraction method would be much more useful if it could be combined with a simple enrichment step. Others have shown that genomic DNA could be captured on silica spin columns from ethanol–NaOH lysis solutions [24]. We found that we could capture DNA from our NaOH lysates using the Mag-Bind RXN Pure Plus, a commercial preparation of paramagnetic beads in unspecified binding buffer. We compared paramagnetic bead purification versus direct dilution for extraction from duplicate samples of broth cultures of three Gram-positive and three Gram-negative bacteria (Figure 3). For these comparisons, the elution from the paramagnetic beads used the same volume as the final volume from direct dilution. The qPCR Ct values for the extractions from the Gram-negative species were nearly equivalent with both methods, while for the Gram-positive, the paramagnetic bead capture gave consistently lower Ct values indicative of reduced PCR inhibition.
The NaOH extraction and magnetic bead capture method was employed on selected air samples from days 42, 44, and 48–56, and all gave positive qPCR-HRM results for 16S rDNA (Table 1), demonstrating that this method is highly effective for extraction from these air samples. The NaOH extraction and magnetic bead capture method was then used for a second BCO challenge experiment in which for days 21–37 we compared computed total CFU to the Ct values for days 21–37 of air sampling (Table 2). The total CFU increases through day 37 and the qPCR Ct values decrease from days 21 to 37 as expected with increasing bacterial load in the air. Therefore, the system we describe using a portable pump and sterile impinger is a low-cost, effective system that can not only collect viable bacteria but also be used to efficiently extract for qPCR analysis of total bacteria or be subjected to species-specific qPCR to detect particular bacteria of interest.

3.3. Optimized Extraction Applied to Diverse Environmental Samples

As NaOH extraction was a reliable method for obtaining bacterial DNA from the air samples, we tested whether this method was also applicable to other environmental samples (Table 3; Figure 4). The bacterial samples include soil; chicken cloacal swabs; food samples; laboratory surfaces; and pond water and sewage. Soil specimens, collected on the University of Arkansas campus, were suspended in 100 mM NaOH with or without 5% saline. The results in Table 3 show that the inclusion of 5% saline during the extraction resulted in increased Ct values (p = 0.02), and purification with paramagnetic beads gives superior amplification compared to direct dilution (p = 0.019). Chicken cloacal swabs, collected at the University of Arkansas Poultry Research farm, were extracted, followed by paramagnetic bead capture or direct dilution. Eight total swabs were individually processed, with four purified by paramagnetic bead capture and four directly diluted for triplicate qPCR-HRM for each swab extracted. Ct values from the paramagnetic purified extracts were lower but not significant (p = 0.13). NaOH extraction was applied to swabs from cheese slices and bread. While none of the swabs from bread gave specific amplification of bacterial 16S, the extracts from swabs from cheese purified with paramagnetic beads gave specific amplification, whereas no amplification resulted from direct dilution. This may result from PCR inhibitors, since alternative methods of DNA extraction from foods with high fat and protein content typically result in the release of significant amounts of PCR inhibitors [38], which may be removed during the paramagnetic bead capture. NaOH extractions from swab samples from four different laboratory surfaces were extracted with 100 mM NaOH followed by either paramagnetic bead capture for two or direct dilution for the other two prior to qPCR-HRM. All four samples gave specific 16S amplification, with paramagnetic bead capture somewhat lower. Sewage and pond water samples collected included the following: (a) incoming sewage from the Paul R. Noland Wastewater Treatment facility, and (b) pond water samples from Clarence Craft Park, both in Fayetteville, AR, USA. Pellets from these samples were resuspended in SPW and brought to 100 mM with NaOH. Separate aliquots of each sample were subjected to either direct dilution or paramagnetic bead capture prior to qPCR-HRM. The Ct values (Figure 4) for the sewage plant effluent were lower than for the pond water samples, and although paramagnetic bead capture gave lower Ct values than direct dilution, that was not always the case.

3.4. Optimized Extraction Use for Fecal Microbiome

The fecal microbiome is an increasingly important focus in domestic animal health and human medicine [39,40,41,42,43]. We compared our NaOH extraction method to a commercial kit for extraction of DNA suitable for PCR amplification of the V4 region of 16S rDNA for microbiome analyses from swine fecal swab samples. The analysis was for six samples including two different growth ages using duplicate swabs for the two extraction methods. Paired swabs were extracted using either a commercial kit or the NaOH extraction with paramagnetic bead capture. Quantification of the PCR products from the NaOH extraction and NanoDrop results from kit extraction are provided in Table 4. The NaOH extraction with magnetic bead capture was comparable, if not superior, to the commercial kit based on the quantification of PCR products (Table 4). The pipeline for analysis of the sequence data (see Methods) has been described [28]. Figure 5 shows the alpha and beta diversity for the swab microbiome. Kruskal–Wallis test results show no significant differences between alpha diversities for the two extraction methods (see p values in Figure 5). Analysis of similarity (ANOSIM) conducted in qiime2 also shows that microbiome communities of these two methods were very similar. The swine gut microbiome structure based on Bray–Curtis distance (Figure 5C) and membership as measured by Jaccard distance (Figure 5D) were separated by animal growth stage for lactation versus nursery (ANOSIM, R = 0.962, p = 0.003 for Bray–Curtis distance; R = 0.998, p = 0.001 for Jaccard distance) but not by DNA extraction methods (ANOSIM, R = −0.081, p = =0.767 for Bray–Curtis distance; R = −0.120, p = 0.832 for Jaccard distance). Figure 6 shows the comparisons of the microbiome composition at the phylum and genus levels for the pairs of swabs from the two ages. Although there are differences between individual pigs for the minor phyla, especially at 10 days of age, the paired samples show similar distributions and frequencies whether extracted with the kit or with the NaOH with paramagnetic bead capture. There are more differences between the extraction methods for the genus-level analyses, but the data clearly support the NaOH extraction with magnetic bead capture as an inexpensive alternative for high-throughput microbiome analyses.

3.5. Evaluation of Other Paramagnetic Beads and Binding Buffer

As the bulk of our paramagnetic bead capture used the Mag-Bind RXN Pure Plus, which consists of paramagnetic beads supplied in a binding buffer, we repeated our NaOH extractions from bacterial culture samples using PureSil-Silica paramagnetic beads and a defined bead-binding buffer (Methods). The average Ct values for the PureSil-Silica were 25.1 ± 0.3, while the Mag-Bind RXN Pure Plus Ct values were 30.1 ± 0.3. Thus, either source can bind DNA, but there may be some differences in either capacity or efficiency of capture under alkaline conditions, with the PureSil beads and defined bead-binding buffer apparently recovering more DNA from equivalent aliquots. Additionally, with the PureSil beads and a separate buffer, we could examine different ratios of binding buffer to NaOH. First, we determined that a 1:1 mixture of 100 mM NaOH and the defined bead-binding buffer is pH 12.9. Therefore, the DNA capture is clearly under high pH. We then examined DNA recovery using ratios of binding buffer to 100 mM NaOH of 0:1, 0.25:1, 0.5:1, 0.75:1, and 1:1, followed by ethanol rinses, elution in Te, and qPCR quantification. While some DNA was captured even at 0:1 (Ct = 33.5 ± 0.1 with specific HRM), increasing binding buffer increased yield: 0.25:1 Ct = 23.5 ± 0.5, 0.5:1 Ct = 21.2 ± 0.2, 0.75:1 Ct = 20.9 ± 2.0, and 1:1 Ct = 20.8 ± 1.0. Size selection of double-stranded fragments was reported through the ratio of binding buffer under neutral pH [44,45]. Given that our quantification is based on qPCR for chromosomal markers, it appears that 0.5:1 binding buffer to 100 mM NaOH is sufficient to capture the larger DNAs in this extraction procedure. For smaller fragments, it may be better to use 1:1 or higher ratios.

4. Discussion

The use of 100 mM NaOH to lyse bacteria and extract bacterial DNA from several different environmental samples presents an inexpensive method. Many of these extracts can be directly subjected to qPCR by fivefold dilution, where the final DNA sample is less than or equal to 1/10th of the qPCR volume. For some samples, the sensitivity is improved through capture of the liberated DNA using silica-coated paramagnetic beads. Shi et al. [46] determined that DNA has two major binding mechanisms for silica at pH > 3: through the phosphate backbone and hydrophobic binding of the DNA bases. Single-stranded nucleic acids bind to silica with greater affinity than double-stranded nucleic acids because the hydrophobic bases are exposed and because single-stranded DNA is more flexible than double-stranded DNA, maximizing contacts. Typically, nucleic acids are driven onto silica using chaotropic salts that dehydrate the nucleic acid and silica, but some amino acid buffers were also demonstrated to comparably facilitate nucleic acid binding to silica [47]. We used a recommended bead-binding buffer (BBB—see Methods) where a 50:50 mixture of 100 mM NaOH and BBB is pH 12.9. Surprisingly, some DNA will bind to the beads in 100 mM NaOH with no addition of BBB. However, more DNA is recovered from bacterial lysates through the addition of at least 0.5:1 BBB to 100 mM NaOH. Presumably, alternative binding buffers could also be compatible for DNA capture from high-pH solutions.
We compared the alkaline lysis method with different rapid methods for DNA extraction from bacterial cells collected from air samples. These included rapid boiling; sonication; bead beating; enzyme digestion; and C2minOAc. The 100 mM NaOH protocol was almost always successful for broth bacterial cultures and somewhat less successful for toothpick samples of bacterial colonies. We previously used a 10 min 100 °C treatment for toothpick samples of bacterial colonies with routine success, but that suffered from over- or undersampling of the colony [26,48]. The 100 mM NaOH extraction seems to be much less susceptible to “over-load”. We also successfully used the 100 mM NaOH extraction protocol to extract DNA from samples from air, garden soil, chicken cloaca, food, environmental surfaces, sewage, pond water, and pig rectal swabs. This technique provides a high degree of reliability, has a short incubation time, and produces readily amplifiable PCR substrates. NaOH stocks are stable at ambient temperature, and the extraction requires no organic solvents, inhibitory compounds, expensive enzymes, or special equipment (shakers, mixers, heaters, etc.). Of note, assessment of an animal microbiome is related to animal nutrition and health and is a research topic of many scientists [28,39,40,49,50,51,52,53]. Efficient DNA extraction from specimens such as fecal swabs is critical to reveal microbial composition and structures. Interestingly, the paired swab microbiomes illustrated by the two DNA extraction methods, a commercial kit and the NaOH extraction, were very similar in terms of community composition and overall alpha and beta diversities. Thus, the inexpensive NaOH–magnetic bead extraction technique could be used to replace the more expensive and time-consuming commercial kit(s) for microbiome studies.
One disadvantage of NaOH DNA extraction might be in converting the dsDNA to ssDNA, which requires special reagents for high-sensitivity quantification by fluorimetry and is problematic for size analysis by standard agarose gel electrophoresis. However, the liberated DNA can be easily quantified using qPCR. The NaOH method of DNA extraction has significant importance when extracting DNA from any bacterial species that cannot be cultivated, as might be present in sampling air, soil, sewage or feces. Therefore, these methods are directly applicable to sampling in resource-constrained environments. The extracted DNA can be utilized for conventional or quantitative PCR with relative ease, and it may also be useful for other molecular diagnostics such as LAMP or nanopore sequencing. NaOH extracts diluted fivefold (with water or with Te) were stored at −20 °C for 30 days with no loss of qPCR sensitivity, so the extracts are stable. Further, the 10 min treatment with 100 mM NaOH is similar to standard plasmid isolations from bacteria, so there is no evidence of depurination or significant DNA fragmentation. Our assays are exclusively for an approximately 900 base pair region of the 16S ribosomal RNA gene, but given that alkaline-extracted plasmids have been efficiently sequenced for many years, we believe there is little concern regarding the length or integrity of the extracted DNAs.
In summary, the liberation of genomic DNA from bacteria using 100 mM NaOH followed by purification using paramagnetic beads or a 1:5 dilution in Te offers many benefits over conventional enzymatic techniques and commercial kits. The proposed technique is distinguished by the fact that it requires as few as one incubation step of ten minutes, and no centrifugation. Even with magnetic bead capture, the method can be completed in less than one hour. We have routinely scaled up the NaOH extraction and magnetic bead capture to process 100 µL of overnight stationary Staphylococcal cultures to produce 1 mL at 10–30 ng/µL, based on ΔΔCt for qPCR vs. control DNA [37], thus avoiding larger culture volumes, multiple centrifugations, long incubation times, expensive enzymes, and organic solvents [14]. This simple extraction method combined with an inexpensive air-sampling system, as we have described, should be readily employed in agricultural systems to detect and monitor bacterial pathogens for rapid response to critical diseases. The method may also be of utility for DNA viruses and fungal pathogens.

Author Contributions

A.S. developed the extraction techniques and wrote the initial draft of the manuscript. B.Z. produced and analyzed the swine microbiome data. A.A., J.Z. and D.D.R. secured funding and oversaw the research. D.D.R. was responsible for the final draft of the manuscript with contributions from A.S., B.Z., A.A. and J.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000, and the Ginny Lewis Fund of the Fulbright College of Arts and Sciences at the University of Arkansas.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare that they have no competing interests. The funders played no role in the design of the experiments, the interpretation of the data, the writing of this manuscript, or the decision to publish this manuscript.

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Applsci 14 00141 g001
Figure 1. Average Ct values for different NaOH concentrations (mM) for extraction from duplicate aliquots (light gray vs. dark gray) of 4 × 103 CFU of an S. agnetis 908 broth culture followed by dilution with 4 volumes of Te and then qPCR-HRM. Data plotted are average for triplicate qPCR. Error bars are standard deviation. Ct values for controls: 0.25 ng 908 DNA—11.8 ± 0.5; H2O—No Ct.
Figure 1. Average Ct values for different NaOH concentrations (mM) for extraction from duplicate aliquots (light gray vs. dark gray) of 4 × 103 CFU of an S. agnetis 908 broth culture followed by dilution with 4 volumes of Te and then qPCR-HRM. Data plotted are average for triplicate qPCR. Error bars are standard deviation. Ct values for controls: 0.25 ng 908 DNA—11.8 ± 0.5; H2O—No Ct.
Applsci 14 00141 g001
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Figure 2. Average Ct values from qPCR-HRM for boil (gray) vs. NaOH extraction (green) for duplicate samples from broth cultures of Staphylococcus agnetis, Enterococcus cecorum, or three isolates of Enterococcus faecalis. Overnight cultures were pelleted and suspended in 10 volumes of sterile water. Duplicate 100 aliquots were treated by boiling (10 min at 100 °C in PCR machine) or NaOH (addition of 11 µL 1 M NaOH for 10 min at room temperature). The samples were then diluted with 400 µL Te and assayed in triplicate by qPCR. Error bars are standard deviation. There was no amplification for E. faecalis 1570 for the boil extract. Ct values for controls: 2 ng 908 DNA—19.6 ± 0.7; H2O—No Ct.
Figure 2. Average Ct values from qPCR-HRM for boil (gray) vs. NaOH extraction (green) for duplicate samples from broth cultures of Staphylococcus agnetis, Enterococcus cecorum, or three isolates of Enterococcus faecalis. Overnight cultures were pelleted and suspended in 10 volumes of sterile water. Duplicate 100 aliquots were treated by boiling (10 min at 100 °C in PCR machine) or NaOH (addition of 11 µL 1 M NaOH for 10 min at room temperature). The samples were then diluted with 400 µL Te and assayed in triplicate by qPCR. Error bars are standard deviation. There was no amplification for E. faecalis 1570 for the boil extract. Ct values for controls: 2 ng 908 DNA—19.6 ± 0.7; H2O—No Ct.
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Figure 3. Average Ct values from qPCR-HRM for NaOH extracts from broth cultures of 3 Gram-positive (Staphylococcus aureus 1516, Staphylococcus agnetis 908, Enterococcus faecalis 1558) and 3 Gram-negative (Escherichia coli 1409, Escherichia fergusonii 1412, Salmonella enterica 1414) species. Extracts were processed by paramagnetic bead capture (open bars) or by dilution with 4 volumes of Te (gray bars). Final samples for each method were adjusted to the same volume and assayed by qPCR-HRM in triplicate. Error bars are standard deviation. Ct values for controls: 2 ng 908 DNA—12.6 ± 1.1; H2O—31.5 ± 1.0 (no melt product for 16S product).
Figure 3. Average Ct values from qPCR-HRM for NaOH extracts from broth cultures of 3 Gram-positive (Staphylococcus aureus 1516, Staphylococcus agnetis 908, Enterococcus faecalis 1558) and 3 Gram-negative (Escherichia coli 1409, Escherichia fergusonii 1412, Salmonella enterica 1414) species. Extracts were processed by paramagnetic bead capture (open bars) or by dilution with 4 volumes of Te (gray bars). Final samples for each method were adjusted to the same volume and assayed by qPCR-HRM in triplicate. Error bars are standard deviation. Ct values for controls: 2 ng 908 DNA—12.6 ± 1.1; H2O—31.5 ± 1.0 (no melt product for 16S product).
Applsci 14 00141 g003
Applsci 14 00141 g004
Figure 4. Average Ct values from qPCR-HRM for NaOH extracts from three samples from sewage water (A–C) or pond water (D–F). Aliquots from each extract were subjected to paramagnetic bead purification (open bars) or dilution with 4 volumes of Te (gray bars). Final samples for each method were adjusted to the same volume and assayed by qPCR-HRM in triplicate. Error bars are standard deviation. Ct values for controls: 0.25 ng 908 DNA—16.1 ± 2.5; H2O—32.4 ± 0.45 (no melt product for 16S product).
Figure 4. Average Ct values from qPCR-HRM for NaOH extracts from three samples from sewage water (A–C) or pond water (D–F). Aliquots from each extract were subjected to paramagnetic bead purification (open bars) or dilution with 4 volumes of Te (gray bars). Final samples for each method were adjusted to the same volume and assayed by qPCR-HRM in triplicate. Error bars are standard deviation. Ct values for controls: 0.25 ng 908 DNA—16.1 ± 2.5; H2O—32.4 ± 0.45 (no melt product for 16S product).
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Figure 5. Microbiome alpha diversity (A,B) and beta diversity (C,D) from six pig fecal samples extracted either with a kit (circle) or the NaOH (triangle) method. Samples numbers are the same as in Table 4, and the paired samples are represented with the same color and connected with a line.
Figure 5. Microbiome alpha diversity (A,B) and beta diversity (C,D) from six pig fecal samples extracted either with a kit (circle) or the NaOH (triangle) method. Samples numbers are the same as in Table 4, and the paired samples are represented with the same color and connected with a line.
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Figure 6. Microbiome composition by phylum (A) or genus (B) from six pig fecal samples extracted either with a kit or the NaOH method. Sample numbers (top of paired bars) are as in Table 4, and the extraction method is indicated at the bottom.
Figure 6. Microbiome composition by phylum (A) or genus (B) from six pig fecal samples extracted either with a kit or the NaOH method. Sample numbers (top of paired bars) are as in Table 4, and the extraction method is indicated at the bottom.
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Table 1. Air samples, bacterial counts, extraction method, and qPCR results during a 56-day lameness trial in spring 2021. Samples were collected using the impinger on the indicated day for a given sampling duration (minutes). For each sample, the resuspended pellet was divided for serial dilution for plate counts and for DNA extraction. Total CFU is the computed total viable bacteria in the original sample. Extraction indicates the method used (see text) for that sample where NaOH + MB is the NaOH extraction with magnetic bead capture and elution. qPCR positive indicates whether that extract produced a specific product for qPCR-HRM, where NS signifies no specific amplification based on HRM. Avg Ct 16S presents the average Ct (±SEM) from triplicate samples using 8F × 936R 16S primers, where ND (not determined) indicates that species-specific qPCR primers were used. All qPCR-HRM assays included positive controls of purified 908 DNA and negative controls of H2O, which performed as expected.
Table 1. Air samples, bacterial counts, extraction method, and qPCR results during a 56-day lameness trial in spring 2021. Samples were collected using the impinger on the indicated day for a given sampling duration (minutes). For each sample, the resuspended pellet was divided for serial dilution for plate counts and for DNA extraction. Total CFU is the computed total viable bacteria in the original sample. Extraction indicates the method used (see text) for that sample where NaOH + MB is the NaOH extraction with magnetic bead capture and elution. qPCR positive indicates whether that extract produced a specific product for qPCR-HRM, where NS signifies no specific amplification based on HRM. Avg Ct 16S presents the average Ct (±SEM) from triplicate samples using 8F × 936R 16S primers, where ND (not determined) indicates that species-specific qPCR primers were used. All qPCR-HRM assays included positive controls of purified 908 DNA and negative controls of H2O, which performed as expected.
DayMinutesTotal CFUExtractionqPCR Positive Avg Ct 16S
1734100BoilYes ND
17108825BoilYes ND
201512,205BoilNS
203021,050BoilNS
21204500Bead beatingNS
272035,200SonicationNS
29204000SonicationNS
352017,200SonicationNS
42206000NaOH + sodium acetateNS
42206600NaOH + MBYes 23.9 ± 0.03
442020,000BoilNS
442029,300NaOH + MBYes 22.0 ± 0.1
462046,400BoilNS
462020,000NaOH + C2mimOAcYes 20.8 ± 0.1
482016,000C2mimOAcNS
482020,000NaOH + MBYes 23.1 ± 0.1
50208000NaOH + MBYes 24.4 ± 0.2
502012,000NaOH + MBYes 20.5 ± 2.4
52209000NaOH + MBYes 23.2 ± 0.6
52205000NaOH + MBYes 23.3 ± 0.6
542012,200NaOH + MBYes 23.2 ± 0.5
542012,500NaOH + MBYes 17.4 ± 2.0
562020,000NaOH + MBYes 19.7 ± 2.4
562052,900NaOH + MBYes 21.8 ± 0.3
Table 2. Air samples, bacterial counts, and qPCR Ct values during a 56-day lameness trial in which all samples were collected in 20 min and extracted using 100 mM NaOH and paramagnetic bead capture. Columns are as in Table 1 except that for the qPCR, which displays the average of triplicate Ct values with SEM. All qPCR-HRM assays included positive controls of purified 908 DNA and negative controls of H2O, which performed as expected.
Table 2. Air samples, bacterial counts, and qPCR Ct values during a 56-day lameness trial in which all samples were collected in 20 min and extracted using 100 mM NaOH and paramagnetic bead capture. Columns are as in Table 1 except that for the qPCR, which displays the average of triplicate Ct values with SEM. All qPCR-HRM assays included positive controls of purified 908 DNA and negative controls of H2O, which performed as expected.
DayTotal CFUAverage Ct ± SEM
2134022.3 ± 0.4
21152020.5 ± 0.2
23126019.5 ± 0.2
23201018.3 ± 0.1
25208016.0 ± 0.01
25364017.4 ± 0.1
27458020.1 ± 0.01
27365019.3 ± 0.01
3711,20016.0 ± 0.1
3712,80015.8 ± 0.02
Table 3. Ct values for qPCR from environmental samples extracted with 100 mM NaOH as in Methods, with MB (magnetic beads) or Dil (direct dilution) prior to qPCR-HRM in triplicate with 16S primers, where NS signifies no specific amplification based on HRM. All qPCR-HRM assays included positive controls of purified 908 DNA and negative controls of H2O, which performed as expected.
Table 3. Ct values for qPCR from environmental samples extracted with 100 mM NaOH as in Methods, with MB (magnetic beads) or Dil (direct dilution) prior to qPCR-HRM in triplicate with 16S primers, where NS signifies no specific amplification based on HRM. All qPCR-HRM assays included positive controls of purified 908 DNA and negative controls of H2O, which performed as expected.
SampleMB/DilqPCR-HRM Ct ± SEM
Soil with 5% salineDil30.4 ± 0.04
MB31.2 ± 0.3
Soil with H2ODil27.5 ± 0.2
MB25.8 ± 0.1
Four chicken cloacal swabsDil21.9 ± 1.4
MB22.7 ± 2.2
CheeseDilNS
MB19.6 ± 0.07
BreadMB/DilNS
Lab surface 1Dil30.4 ± 0.1
Lab surface 2MB26.6 ± 0.3
Lab surface 3Dil32.3 ± 0.4
Lab surface 4MB29.8 ± 0.4
Table 4. DNA quantification from duplicate fecal swabs from each of six pigs of two growth stages, day 10 lactation or day 59 end of nursery. Pairs of swabs were extracted either with a commercial kit (Kit) or the NaOH extraction with magnetic bead capture (NaOH + MB). NaOH extraction was quantified by qPCR; kit extraction was quantified with a NanoDrop.
Table 4. DNA quantification from duplicate fecal swabs from each of six pigs of two growth stages, day 10 lactation or day 59 end of nursery. Pairs of swabs were extracted either with a commercial kit (Kit) or the NaOH extraction with magnetic bead capture (NaOH + MB). NaOH extraction was quantified by qPCR; kit extraction was quantified with a NanoDrop.
IDPig Age (Day)PCR Product (ng/µL)
KitNaOH + MB
11024127
210685739
31022410
4595552
55982216
65994150
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Shwani, A.; Zuo, B.; Alrubaye, A.; Zhao, J.; Rhoads, D.D. A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses. Appl. Sci. 2024, 14, 141. https://doi.org/10.3390/app14010141

AMA Style

Shwani A, Zuo B, Alrubaye A, Zhao J, Rhoads DD. A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses. Applied Sciences. 2024; 14(1):141. https://doi.org/10.3390/app14010141

Chicago/Turabian Style

Shwani, Abdulkarim, Bin Zuo, Adnan Alrubaye, Jiangchao Zhao, and Douglas D. Rhoads. 2024. "A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses" Applied Sciences 14, no. 1: 141. https://doi.org/10.3390/app14010141

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