Development of Nested PCR for SARS-CoV-2 Detection and Its Application for Diagnosis of Active Infection in Cats
, Ralitsa Popova-Ilinkina 2, Dobrinka Ivanova 1,3, Nikolina Rusenova 4, Hristiyan Mladenov 5, Kalina Mihova 6 and Ivan Mitov 1
Round 1
Reviewer 1 Report
The paper of Sirakov et al. Describes nested PCR method for detection of SARS-CoV2 in human and cat samples. As in the course of COVID-19 pandemy plenty of NAT methods have been developed for SARS-CoV2 diagnosis and many of them are validated for IVD, this paper is only of moderate importance. Its main contribution is validation of the method described for SARS-CoV2 diagnostics in domestic cats. However, some sentences in both the Materials and Methods a Results paragraphs are not clearly formulated and need amendment or modification. Moreover, it is not documented, whether the method is able to detect all the SARS-CoV2 variants.
Main comments:
Materials and methods:
Line 68-69: It is not clear what kind of material was analysed repeatedly: The samples, RNA or cDNA extracts prepared from them? If the first is correct, how the samples were stored before the second analysis? Were there aliquoted or repeatedly thawed?
Results:
The sentence 128-130 should be re-formulated: Positivity of 31/45 samples does not give 100%. Which „other“ samples? The samples negative in the first run of nested PCR?
Lines 136-138: How can be the concentration 45.5 ng/µL reached from 5.7 ng/µL of the reference strain RNA?
Discussion:
Line 171: Dilution 107 does not tell anything as for the sensitivity of the assay.
The sensitivity of the nested PCR for currently prevailing SARS-CoV2 variants should be documented.
Minor comments:
Materials and methods:
Explain abbreviations FHV,FCV. The methods used for characterization of FNV, FCV or FCoV positivity should be cited.
Line 74: What does 109 in the reference strain mean? Genome copies or the virus particles?
Results:
Line 114-115: The sentence should be divided into two: RNA extracts were probably analysed by NanoDrop, while PCR products by gel electrophoresis ( which was used for quantitation of the reference SARS-CoV2 strain, while in other PCR products it was qualitative only).
Discussion:
Line 188: ...complementary DNA....
Which countermeasures were used to prevent false positive results due to cross-contamination?
Author Response
Please, see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Summary:
Authors designed a nested PCR based detection assay for SARS-CoV2 and tested it on human and cat samples. Test appears efficient and specific. Overall, the manuscript is straightforward and written accordingly. However, there is a clear lack of rigor in preparing the figures. Please improve visibility and presentation to reach publication standards.
Major comments:
- Lines 62-65: The authors used 90 patient samples total. It is important to mention approval of institute (bio)ethics committee if needed. What is the nature of the samples?
- Lines 73-74: That sentence is poorly written making it hard to understand what the authors used as control exactly.
- Acronyms need to be defined at their first used. Many such as (IVD, LAMP, FHV, FCOV).
- Figure 1.B: Figure legend need improving. Right now, it is very confusing. Please make sure to add visual cues to increase visibility such as:
- Do not reset lane number from lower part,
- Mark ext/Int primer PCR products,
- Visual gradient/increase of RNA concentration.
- What is the purpose of figure 2? Right now, with the current version of the manuscript it Is very unclear what the authors are trying to argue.
Author Response
Please, see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors have updated the manuscript and addressed my comments.
