A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses
, Jiangchao Zhao 1,2 and Douglas D. Rhoads 1,4,*Round 1
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsIn my impression, there are many reports about DNA extraction with NaOH solution. To this end, I searched in the database and found that the paper "A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples" published in Mol Biochem Parasitol (doi: 10.1016/j.molbiopara.2023.111557) provided a fast, simple, easy, efficient, and inexpensive method for DNA extraction. Notably, this method utilized 10-50mM NaOH at room temperature to isolate DNA. However, authors in this resubmitted MS emphasized that "our search of the literature could not find one example where NaOH at room temperature alone was used." Accordingly, I'll stick with my idea that the MS seems be deficient in the novelty of the methods, and it is not suitable for publication in Applied Sciences.
Author Response
Response: the manuscript suggested does use NaOH for lysis but this is for protozoa, and so we should distinguish our approach by restricting it to bacterial samples, which was our goal and the only organisms we investigated. Interestingly, the method presented in the Plasmodium paper only appears to lyse the protozoan but not really “extract” the DNA since the DNA remains on the filter paper. That seems very strange that the paper punch is treated with 0-50 mM for 20 minutes, then washed with TE buffer for 2 min, and then the paper punch is air dried and used for PCR. It seems that NaOH is not required based on their data, and the DNA is not extracted but it is amenable to PCR amplification while still “stuck” to the filter paper. Is that really extraction? Given that even 0 mM NaOH worked at higher infection levels and the DNA becomes exposed but somehow still bound to the filter paper is surprising. Indeed, even simple salt solutions can be used to “liberate” human cell DNA. Never-the-less, we have modified the manuscript to specifically include only bacterial samples as targets.
Reviewer 2 Report (Previous Reviewer 1)
Comments and Suggestions for AuthorsThis reviewer believes that the manuscript has been improved by the authors and now warrants publication in "Appl. Sciences" journal.
Author Response
Response: we thank the reviewer for their support.
Reviewer 3 Report (New Reviewer)
Comments and Suggestions for AuthorsThe authors established a DNA extraction method based on sodium hydroxide lysis. This method is simple, fast and has been confirmed in a variety of samples.
Through the trial of different sodium hydroxide concentrations, the recommended concentration of this method was explored. The experimental design is reasonable and the experimental results can demonstrate the experimental conclusions.
Will sodium hydroxide cause damage to DNA and affect subsequent analysis? Please add analysis of DNA fragment size to the experimental method or discuss this possibility in the discussion and the subsequent problems it may bring.
Author Response
Response: agarose gel electrophoresis would not be diagnostic because the DNA has been denatured. At neutral pH we would expect it to form hairpins and snapback DNA which would not be diagnostic of the actual fragment sizes. Given that NaOH lysis has been used for isolation of plasmid DNAs for decades where plasmids of 40 kbp (cosmids) are routinely purified where bacterial chromosomes are fragmented due to shear forces and denatured. The Isch Horowitz method is based on the fragility of the larger DNAs. Given that the plasmid and cosmid DNAs purified using 150 mM NaOH at room temperature (or 37 oC) are amenable to direct DNA sequencing using Oxford Nanopore long reads supports that no significant changes occur in the DNA sequences. Further, DNA denaturation with alkali is used for Southern blots with no significant degradation of the DNA (size, depurination, etc.). Granted that the majority of our assays are for a 928 bp region of 16S rRNA gene we can not speak to whether there has been some reduction in overall length. However, we have added some additional text in the discussion to clarify what we do and do not know.
Round 2
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsThe quality of this revised manuscript has improved significantly. The following specific comments should be resolved to make the revised manuscript is suitable for publication.
1. It is necessary to systematically evaluate this nucleic acid extraction method, especially the detection sensitivity of this method. It is strongly recommended to systematically compare the NaOH mediated method with other classical or traditional methods including boiling and commercial kit. Therefore, the sensitivity data of the used method should be performed.
2. Increase the statistical difference analysis of data.
3. Figure 1 should increase positive controlsuch as commercial kit method and negative control results.
4. Figure 2, the Ct value of E. faecalis 1570 should be displayed. Ct value should be 35 or 40. In fact, authors should set the cycle parameters as following: 90°C for 45 seconds, 40 cycles of 90 °C for 15 sec, 71.5 °C for 15 sec, and 72 °C for 60 seconds with a plate read after each cycle. The advantage of such parameter setting is that the Ct value is objective data rather than the current Ct value should add 5 to get the actual Ct value which will mislead readers or reviewers.
Author Response
The quality of this revised manuscript has improved significantly. The following specific comments should be resolved to make the revised manuscript is suitable for publication.
- It is necessary to systematically evaluate this nucleic acid extraction method, especially the detection sensitivity of this method. It is strongly recommended to systematically compare the NaOH mediated method with other classical or traditional methods including boiling and commercial kit. Therefore, the sensitivity data of the used method should be performed.
Response: We have already compared the NaOH method to standard lysis methods for the air samples in Table 1 where we tested several different methods for rapid lysis for rapid qPCR assays. In Table 1 we did not try commercial kits because most kits require several hours and are far too expensive for routine testing. In Figure 2 we did direct comparisons of a generally accepted boil method and our NaOH method showing the enhanced performance of the NaOH method. In the Microbiome work in Table 4 and Figures 6 and 7 we compare a commercial kit to our NaOH+MB method demonstrating comparable, if not superior, performance of the NaOH+MB method. The other figures demonstrate that the NaOH method (with or without MB) are useful in different environmental samplings, demonstrating the potential for widespread use.
- Increase the statistical difference analysis of data.
Response: We have already provided averages with either standard deviation or standard error of the means. We are not certain what additional statistical analyses the reviewer expects.
- Figure 1 should increase positive controlsuch as commercial kit method and negative control results.
Response: Figure 1 is about determining the approximate minimum concentration of NaOH for routine lysis of a broth culture. In Table 1 we compared different rapid lysis methods for bacteria collected in air samples.
- Figure 2, the Ct value of faecalis1570 should be displayed. Ct value should be 35 or 40. In fact, authors should set the cycle parameters as following: 90°C for 45 seconds, 40 cycles of 90 °C for 15 sec, 71.5 °C for 15 sec, and 72 °C for 60 seconds with a plate read after each cycle. The advantage of such parameter setting is that the Ct value is objective data rather than the current Ct value should add 5 to get the actual Ct value which will mislead readers or reviewers.
Response: The E. faecalis 1570 sample in question did NOT have a Ct value. There was no amplification and there was no HRM peak. Although sometimes we do see late amplification in the negative controls that has no 16S HRM peak, in many of our experiments there is no Ct value for the H2O negative control. To address this we have added “No Ct” labels in Figures 2 and 3. We also added Ct values for the positive and negative controls in the legends for Figures 1-5 and additional explanations in the legends to the tables. In our experiments we always include a positive control (purified S. agnetis 908 DNA) and a negative water control. Our approach is that all qPCR values are “relative” just as in any ΔΔCt experiment where everything is relative to a reference, in our case the the positive control purified DNA. The CFX instrument that we use measures relative fluorescence units (RFU) and routinely has signal noise for some wells during the first 5 cycles which will confound the Ct analysis of the CFX software. That is why we do not record the first 5 cycles (as stated in the methods section). We are confident that the data we present is a valid representation, and it would not be informative or change the results to repeat all the experiments. Given our description of our methods then if people wish to compare their results to ours, the details are all there.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments Regarding the Manuscript:
The authors of the document entitled “A simple inexpensive alkaline method for DNA extraction from environmental samples for PCR surveillance” shown the potential of a simplified NaOH traditional method for DNA bacterial extraction. The authors exposed that this simple alkaline protocol could be a putative substitute for expensive kits for microbial monitoring response during critical time of diseases. The document was written in a manner that is easy to read, even if at the moments the authors are very repetitive for some concepts (example: pp 1 L 2; pp 2, L 61; pp 2, L 379; pp 15, L 416; pp 16, L 465). On the other hand, the references used by the author are too old, the authors must update the sections of the text taking in consideration the data from recent papers. The methods and the experimental procedures are described in sufficient detail to enable future researchers to follow-up conclusions of this work. The results are presented in a clear manner using graphical and tabular means, but this reviewer could not find the statistical significance between samples (fig. 2) or similar evaluated volumes (fig. 3). Despite the minor significance of the obtained results, these were interpreted and discussed with due reference to previously published studies. Nevertheless, the authors are mixing the contents between of Results and Discussion sections, the document should be modified in this aspect to facilitated its reading and comprehension. Although the paper is quite well written and presented, it does contain a few typographical and other errors which may need to be addressed by the authors before the manuscript is deemed acceptable for publication in “Applied Sciences” journal.
Major points:
Page 1 Line 14: This reviewer does not agree with the sentence “can be completed in 10 minutes at room temperature” because in materials and methods (page 3 line 114-140) for some samples the NaOH protocol require between 15 and 20 minutes.
Page 1 Line 37: The reference 32 must be enclosed at the introduction.
Page 3 Table 1; Page 10 Table 2: The authors should edit this table to facilitate its reading. The authors must explain why they developed the UFC data for each sample. This reviewer has some problems to validate the quality of extracted DNA with the alkaline proposed protocol, because the column “qPCR positive” shows No Specific amplifications. That concept is as well mention by the authors (pp 5, Line 212-215). This reviewer has thought that is in contradiction with the main conclusion of this manuscript.
Page 9 Line 283: This reviewer has thought that the use of Mag-Bind RXN Pure Plus not only enriched the simple in DNA, as well improve the quality of it, as is indicated in the name, so the template DNA has less PCR-enzyme inhibitors at the resuspended DNA. And what about the time-consuming?
Page 15 Line 442-444: The authors must refer to the pictures that support this conclusion at the end of this sentence. For this reviewer, the document does not have benchmark data to support the reproducibility concept by this protocol, which is not so low-cost (pp 1, L 14; pp 10, L 308) to extract good quality DNA as the authors advocated.
Minor points:
Page 1 Line 3: Title “PCR surveillance.” must be changed by “PCR surveillance”
Page 1 Line25: Change “Introduction.” to “Introduction”, comment valid for the others titles.
Page 3 Line 96: Change “glycerol.Media included” for “glycerol. Media included”
Page 5 Line 182: Change the name “Te” to “TE buffer”
End of review
Comments on the Quality of English LanguageThis reviewer does not have any comment in this regard.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe research devised a method to quickly extract genomic DNA from environmental samples based on sodium hydroxide lysis of cells with or without capture by magnetic beads for subsequent PCR or quantitative PCR. This simple extraction method combined with an inexpensive air-sampling system might be employed in agricultural systems to detect and monitor viral, bacterial, and fungal pathogens for rapid response to critical diseases. The findings of this study seem to provide new insights into the DNA extraction methods. However, it is well-known that the NaOH is capable of effectively lysing a variety of cell types from diverse biological materials within 10 minutes. Accordingly, the MS seems be deficient in the novelty of the methods, the current version is not suitable for publication in Applied Sciences.
My specific comments are as follows:
1. Table1 misses details about the experiment, including how to suspend the bacteria cells, the volumes of solution or the bacterial density. Why the bacteria samples collected in Day 20 could not be detected while the bacteria cell numbers were only 2-3 times that of Day 17? As the NaOH lysis of cells with or without capture by magnetic beads is the key to the novelty of the article, however, table1 as well as other experimental designs lack of such comparative experiments.
2. The combination of NaOH+MB seems to be also expensive or time-consuming, labor-intensive, and expensive. Therefore, comparative experiments, NaOH VS NaOH+MG, must be conducted to draw a conclusion, which seems to be a significant deficiency in the experimental design.
3. Lines 116-117, “Pellets were re-suspended in 100µl of SPW then mixed with 10 µl 1 M NaOH.” In this case, the NaOH concentration is no longer 0.1M, which is different with other groups. Authors should check it.
4. Line 144, “,” should be “.”
5. It seems irrational to locate the Lines 177-180 in Section 2.6. Please verify.
6. Lines 238-242, to reduce the interference of NaOH on PCR system, the extracted DNA templates were diluted for about 50 times. Actually, authors should mention that the dilution of the solution will reduce the sensitivity of detection, which is a disadvantage of this method.
7. Lines 250-251, 299-310, the font format and size should be uniformed.
8. In Figure 2 and Figure 5, how many bacteria cells are used for detection?
9. The Ct value of qPCR in Figure 2 is merely 12-17(NaOH+MG) or 13-20(diluted NaOH). Generally speaking, such a small Ct value doesn't seem reasonable. Similarly, the Ct value is only 22.3 when the total CFU is 340, viz. the Ct value is about 30-31 when the CFU is only 1 through the theoretical calculation. It is impossible in the clinical detection.
10. Abbreviations should be full names when they first appear, such as MB, C2mimOAc, etc.
