Development of a Real-Time PCR Method for the Detection of European and Siberian Subtypes of Tick-Borne Encephalitis Virus

Round 1

Reviewer 1 Report

The manuscript "Development of a real-time PCR method for detection of European and Siberian subtype of tick-borne encephalitis virus" is well designed study with well described methods and adequate supporting data to justify the RT-PCR assay to detect strains of TBEV.

Author Response

please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this study, a real-time (RT)-PCR method was developed for the detection of European subtypes of TBEV. The design was chosen so that, in addition to PCR specificity, further identification by DNA sequencing can be performed.

The results are described in great detail but some information regarding the methods is missing. The introduction, and the discussion are written in an understandable way.

 

Major point:

Sorry, I am a little confused. Isn't it RT-PCR that needs to be developed to detect viral RNA from flaviviruses? So, the title and text should be checked to that regard. It should also be explained which primers are responsible for reverse transcription. The RT-PCR procedure is not described correctly, and it is not clear if it is a 1-step or 2-step RT-PCR and how many viral input was used. The exact name of the enzymes and the concentrations used needs to be mentioned.
The same applies to the control oligonucleotides used. All information on this is missing.

Minor points:

99: the use of dark green complicates the readability of the printout. Please change the dark colors in other places as well.

151: the name is = F.X. Heinz

158: Sequence identity is unclear. Please provide the sequence of the 450 bp fragment in the appendix section and indicate the positions described.

174/262:  Li/NOR: Please provide the full name of the abbreviation.

180: Please give more information to the controls used. Was it RNA-virus supernatant or …..

195: What are the oligonucleotides representing 5 TBEV variants?

197: is it:  Each peak of the curves represents the TBS-Fa/TBEV oligonucleotide melting temperature (Tm) for five different TBE variants.

198: Please indicate the sequence of the control oligonucleotides used.

215: To make it more clear please indicate the name of the primers  …. Regarding the forward primer xy we were able to…. 

218, 219: “forward primer” can be replaced by its name

225: The target sequences of (primer name) were more ….

242: Please indicate the sequence of the control oligonucleotides used. Please lighten colors for better readability.

286: …together with (primer names) was tested …..

362: Please indicate which TBEV oligonucleotides were used for Tm determination.

Author Response

please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have responded to all questions.