Optimizing Release of Nucleic Acids of African Swine Fever Virus and Influenza A Virus from FTA Cards
Round 1
Reviewer 1 Report
Overall, the study was well designed and the manuscript is well written and presented. There does appear to be a minor (although not statistically significant ) increased in RNA yield when performing an additional extraction step for RNA pathogens (based on this single RNA pathogen example), but not for DNA pathogens. It is probably worth mentioning that this study is using two pathogens to make a generalized assumption about RNA/DNA pathogens. a single clarification or mention throughout the document that the differences identified are not statistically significant. It was unclear in the methods exactly which numbers (Ct value calculated NA yield) and datasets (comparison between methods holding cards constant) were compared in these t-tests. Clarity there and thinking about the potential to compare other data sets (all direct PCR values v all secondary extraction values) to make sure comparisons are thorough and statistically sound could make this a slightly more robust ms, as could the framing of the conclusions.
Specific comments
Lines 14-15: Abstract – Stating “substantially higher” borders on claiming statistical significance. There are lower Ct values for methods using the secondary extraction step; however I do not think a statistical test of comparison between direct pcr and extraction then PCR was conducted across all assay/card combinations (see request in statistical analysis methods section). Multiple t test appeared to test if there was any statistically significant difference between all 11 methods and there was not
Line 94: Dann extraction typo
Line 162: suggest changing to “smallest” Delta Ct value
Line 215-216: sentence beginning with surprisingly. It is worth noting that no statistically significant differences were recorded between direct pcr and additional extractions for either DNA or RNA. It might be acceptable to say that decreases in Ct valued were more notable for the RNA assay
Line 232 – the best results are still the best, but again, there were not any that were statistically significantly better.
Line 244-245: suggest saying “less than 5% of the original sample, assuming even absorbption across surface area”
Line 246: this is not a 20th part of a genome, but again less than 5% the original sample volume or <5% the viral quantity, which would result in an estimated Ct value increase of ~4.3 cycles. Do you have a citation for this value?
Line 356: lower case “o” in complete
Line 394: Is the protocol previously published or similar to a previously published protocol that can be referenced?
Line 421: it appears total reaction volume is 12.25, but my math could be wrong
Lines 446-452 Statistical analysis section:
In this section, please state each comparison type that was made.
were ct values or estimated viral loads compared between extraction types holding the card used constant?
When comparing cards against eachother, were extraction types held constant?
Also, I believe the graphpad multiple t-tests option provides a p-value for each comparison, card 1 v card 2 card 1 v card 3, etc. If so, please report these values in a supplemental table?
Conclusion section:
the direct PCR ct values (or quantified DNA amounts – whichever data were compared) were moderately (not statistically significant) lower (ct values) than samples that underwent a separate extraction. Given the lack of improvement with a second step, the more efficient lab process of direct PCR is sufficient and additional steps not warranted for general use.
for the IAV RNA evaluation, increased sensitivity (although not statistically significant) was noted after performing an additional nucleic acid extraction. For this reason depending on the downstream analyses, a secondary extraction step may be warranted....
In general, most samples aren't diluted when running diagnostic assays, although only an aliquot (perhaps similar to the hole punch being <5% of the surface area) is taken of the extracted DNA
The authors provided information on Ct values from the original sample, as well as the dilution series. Was the Ct value of a non-diluted sample placed on cards recorded? Having information showing the difference in standard sample PCR, as well as undiluted sample via the carts would help readers understand the potential implications of using this technique. This information along with pathogen specific questions, such as “What is a typical viral load?” would help readers utilize this document to adjust/optimize workflows in their labs.
This would help people understand how many real life samples would be missed if the direct PCR method was used compared to adding the RNA extraction step?
A scientist could elect to use card type 1-3 with direct PCR and still not miss any at the 10^-2 dilution... based on these data.
Framing the conclusions in this manner might make the publication more impactful.
Supplemental table 3: what is the significance of the color coding in the mean delta Ct value column? Green, orange, red
Author Response
please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript by Elnagar et al. describes the use of seven FTA cards to simplify the collection, transport, and storage of biological sample fluids by comparing the yield and quality of DNA and RNA using different releasing/extraction methods. For their study dilution series of African swine fever virus and Influenza A virus as surrogates for DNA and RNA viruses are evaluated. The study indicates that direct PCR amplification without additional nuclei acid extraction could be suitable for ASF DNA, whereas for IAV RNA loads can be amplified from FTA cards if a standard procedure including a lysis step is applied. The authors concluded that the usage of optimized protocols to release nuclei acid from FTA cards could improve the recovery of viral genomes of both viruses, delivered best results and can be used as a universal method for viral DNA and RNA.
The evaluation of FTA cards in combination of rt-PCR is a significant advance to expand diagnosis methods, due to the simplification for cold chain and advantage to inactivate pathogens and prevent degradation, along with the capability to collect samples in the field with minimal resources. The manuscript is well written, and length and figures seem appropriated.
Reviewer's comments:
Line 300: The authors stated that samples were spotted on each FTA card and left 48 hours to be dried. Can the authors expand if any experiments on infectivity for ASF or IAV at48 hours have been done to demonstrate that the virus is inactivated completely? It will be of interest to indicate if 48 hours is the time required for the FTA card inoculated with ASF or IAV to be dried in order not to yield replicating virus, and to be safe for transport and shipment through regular post service as indicated on the introduction.
All samples have been tested as dilution -1 to dilution -4. Considering the loss on analytical sensitivity, which is higher for RNA than DNA, will be of interest to add some data using undiluted samples mimicking the collection of samples as perform in the field when resources to prepare dilutions may not be available, and to be sure no inhibition on PCR when samples are used undiluted.
In the study only one ASFV strain and IAV virus were tested. Are the FTA cards and different methods (eluates) able to detect different strains, for example high or low virulent strains, with the same sensitivity as demonstrated for the strains used in this manuscript?
The authors concluded that although no significant differences for all tested releasing methods can be ascertained, the Chelex® 100 Resin methods (M6-E2 and M7-E2) provided excellent results for DNA / RNA releasing. It will be of interest to know if this method performs similarly when other ASF or IAV strains are use.
Additionally, they concluded that the application of Chelex® Resin 100 buffer mixed with 1x Tris EDTA buffer or with TED 22 10 delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards, the impact of this statement would improve by including other DNA / RNA viruses.
It is common that field samples are mishandled and not the same as experimental samples, consequently DNA / RNA quality could change the results. This limitation should be discussed.
Minor comments:
Line 113: “Three further methods…” only 2 methods are listed on parenthesis.
Line 219: remove parenthesis
Line 356: “cOmplete” fix typo to “complete”
Author Response
please see the attachment
Author Response File: Author Response.pdf
