Effect of the Matrix and Target on the Accurate Quantification of Genomic and Plasmid DNA by Digital Polymerase Chain Reaction

Round 1

Reviewer 1 Report

The manuscript “Effect of the matrix and target on the accurate quantification of genomic and plasmid DNA by digital polymerase chain reaction” represents a good piece of work where the digital PCR (dPCR) assays targeting the 11 commonly employed screening elements have been reported.

The manuscript needs to be revised as follows:

·         The CaMV and FMV needs to be in italics as CaMV and FMV.

·         A brief about the SDrice and SDrapeseed needs to be added in the Introduction, and the construct details of these lines need to be added in the Materials and Methods in Section 2.1.

·         A table needs to be added for comparison of dPCR and real-time PCR assays conducted in this study in terms of specificity, limit of quantification, limit of detection,  efficiency, accuracy and precision, cost, ease of use etc. in the Results.

·       In the results, authors have depicted results only in the form of Tables. The profiles of real-time PCR and dPCR needs to be added for atleast 4 targets in the Results in the main text of the manuscript. For rest of the targets, same may be added in Supplementary information.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The reviewed manuscript is dedicated to the comparison of genomic and plasmid DNA for GM-content quantification. Currently, plasmid DNA is often used as a standard to quantify DNA concentration in qPCR and dPCR experiments, including GM-analysis. However, the efficacy of PCR with plasmid and genomic DNA could be different and the difference could affect the results of GM-analysis. Here, the authors compared in ddPCR standards made from plasmid and genomic DNA. The results presented in the manuscript are timely and interesting for scientists in the field of GM production and control. However, several comments need to be made and addressed. 

 

Major issues:

 

1.      Multiplexing of different targets in a single dPCR reaction would decrease technical errors, was this possibility considered? Thus, references loci could be duplexed by using a HEX-channel.

2.      Page 4 line 190-192. “For example, FMV35-QF/R/P of P-FMV35, P-nos-F1/R/Tm of P-NOS, NOS-QF/R/P of T-NOS, and NPTII-F1/R1/P1 of NPTII showed accurate pDNA quantitative results but significantly smaller gDNA quantitative results, Table S2 — pFMV35S (210 bp), P-NOS (94), T-NOS (165), etc.” – longer amplicons produced biased ratio on genomic DNA, which is more plausible explanation than the structure of the genomic DNA as it is indicated on the page 5, lines 204-206. It would be more accurate to use amplicons with the same lengths as it was indicated in precious papers about DNA quantification with ddPCR. Biased pCaMV35S could be the result of uneven GC-content.

3.      Page 5, lines 218-219. “The constructed standard curves using different calibrator types are shown in Table S4” — authors are encouraged to provide corresponding amplification plots in the supplementary file.

4.      “3.2. Quantification of low-level samples by qPCR” — was only T-NOS used to quantify DNA, were the same results obtained for other targets? Authors are encouraged to provide data on accuracy of qPCR analysis with other targets as it was made for ddPCR assays.

5.      Page 5, lines 229-234. “We speculate that the simpler the template structure, the more efficient it is to match the primer/probe in the annealing stage, which thus generates relatively small Ct values. The difference in structural complexity is speculated to be the cause of the matrix matching error, which led to a lack of commutability of the gDNA calibrator to the pDNA calibrator on the qPCR platform.” — the reason behind the obtained results could be less effective amplification of supercoiled plasmid DNA in qPCR. It is well known fact that leads to the apparent more effective amplification of genomic DNA in comparison to plasmid DNA. In the manuscript, plasmid standards were not linearized before the preparation of calibrators, which could lead to the observed overestimation of standard samples. Authors are encouraged to compare linearized plasmid samples with intact plasmid DNA in qPCR for testing this hypothesis.

6.       

 

Minor issues:

 

1.      Page 1, lines 22-24. “The accuracy of the qPCR quantification of the low-level pDNA and gDNA test samples was low when pDNA was used as a calibrator, that of the dPCR quantification was high when the screening elements were used as targets, and not affected by variations in template type and detection target.” — the sentence is confusing, please, reformulate.

2.      Matrix instead of more commonly used template

3.      Page 1, lines 72-73. “Whether the selection of a detection target affects the characterization result of multiple-target RMs by dPCR has not been determined.” — the sentence is confusing, please, reformulate.

4.      Page 2 line 62. “plasmid was found on the Fluidigm dPCR platform“ – the effect of plasmid conformation was noted for Bio-Rad dPCR platform (10.3389/fmolb.2020.00155, 10.1007/s00216-013-7546-1).

5.      Page 3 line 98. “adjusted to approximately 50 ng/μL using 0.1×TE buffer“ — was a carrier used to avoid DNA precipitation on tube walls?

6.      Page 3 lines 137-139. “The qPCR assays were performed on a CFX384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) in a final volume of 10 μL that contained 1 μL gDNA, 1 × TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 0.4 μL of each primer (10 μM), and 0.2 μL of probe (10 μM).” – small volumes less than 1 μL would increase a pipetting error, was there a possibility to avoid it?

7.      Page 4 lines 148-149. “using dPCR Supermix for Probes kit (Bio-Rad, Pleasanton, CA), with the same final concentration of both the primer and probe as that of qPCR in 10 μL of the mixture” – the concentration of primers/probes of 900/250 nM is standard in ddPCR, why was 400/200 nM used instead of it?

8.      Page 4 line 154. “50 cycles of 94°C for 30 sec and 60°C for 1 min” – was elongation temperature gradient performed for primers/probes sets used in the study?

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Very interesting and relevant study, providing data on the use of plasmid DNA as calibrators. I have one comment on the results: in part 3.2 the authors suggest that the complexity of the matrix (plasmid or genomic DNA) can be reason for the Ct variation. Some other causes can be also investigated: inhibition effect or variation due to the low copy number of the target in case it is below the quantification limit of the method. It would be beneficial to comment on that. 

Author Response

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Round 2

Reviewer 1 Report

Most of the suggestions have been incorporated.

Author Response

No further comment needs response.

Reviewer 2 Report

Authors have scrupulously and carefully edited the manuscript, and mentioned questions are solved; all statements and changes are supported by the results and cited articles. Many thanks to authors for their efforts and attention to all remarks from the review. However, a few comments need to be made and addressed. 

 

Minor issues:

 

1.      Usually, a term “template” is used to refer a DNA amplifying in PCR, when “matrix” normally means a material and structures between eukaryotic cells. Using “matrix” instead of “template” could be confusing for readers.

2.      “At the beginning, we carried out the primer/probe concentration testing. The dPCR can obtain expected performance by testing the concentration of 400/200 nM, therefore this concentration was used.” — would it be possible to include the results of the comparison in the manuscript or in the supplementary file? Concentration of primers and probes strongly affects the results of ddPCR, as it defines the distance between positive and negative clusters. Therefore,

3.      “no elongation temperature gradient was set in this study” — elongation temperature also affects the efficacy of both qPCR and ddPCR. Plausibly, the difference between various primers sets could be caused by suboptimal elongation temperature, which needs to be mentioned in the manuscript.

Author Response

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Author Response File: Author Response.pdf