Two TaqMan real-time quantitative PCR assays for the detection of Alongshan virus, a new member of the tick-borne Flaviviridae family
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Introduction. Alongshan virus (ALSV) is a tick-borne Flaviviridae . It has been detected in Ixodes ricinus and Ixodes persulcatus across China, Russia, Finland, Switzerland and Germany.
Hypothesis/Gap Statement. However, the clinical relevance and the pathogenicity of ALSV in humans remain unclear. Sensitive and specific molecular tools are needed to support surveillance and to enable clinical investigations of ALSV in suspected cases of tick-borne meningoencephalitis.
Aim. We aimed to develop, validate and integrate two ALSV-specific TaqMan real-time quantitative PCR (qPCR) assays on our open, high-throughput molecular diagnostic platform.
Methodology. We designed assays targeting conserved regions of the NS3 (helicase-protease) and NS5 (RNA-dependent RNA polymerase) genes, incorporating degenerate bases and locked nucleic acid modifications where needed to accommodate documented viral diversity and to harmonize the annealing temperature with TaqMan probe-related technologies and our platform. Analytical sensitivity and reproducibility were assessed using synthetic plasmids carrying the targets; specificity was evaluated against 41 cerebrospinal fluid (CSF) pathogens and 30 winter CSF specimens from patients with suspected central nervous system infection. ALSV-positive Swiss tick extracts served as biological positives.
Results. Detection frequencies for NS3 PCR were 100%, 100%, 92%, 72%, 20%, 28 and 0% at 1,000, 100, 10, 5, 2, 1 and 0.1 copies per reaction, respectively. For NS5, the detection frequencies were 100%, 100%, 92%, 88%, 40%, 20% and 0% at the same concentrations. Using a priori definition of limit of detection (LoD) as ≥95% positive replicates, LoD was 100 copies per reaction for both real-time qPCRs. However, as the PCRs are performed in triplicate in our platform, the LoD can be estimated at five copies per reaction for the NS3 real-time qPCR and two copies per reaction for the NS5 PCR. Intra- and inter-run reproducibility across five independent runs met diagnostic standards. Specificity was 100% (71/71). ALSV-positive tick samples were detected by both assays, with lower cycle thresholds for NS5.
Conclusions. We validated two ALSV real-time qPCR assays suitable for integration into open molecular diagnostic platforms. These assays enable syndromic testing alongside other encephalitis-associated viruses (e.g., Tick-borne encephalitis virus and West Nile virus) and will facilitate timely clinical management of suspected cases, high-throughput tick surveillance and future clinical studies of potential ALSV pathogenic role.
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I am pleased to inform you that, after careful consideration of your revisions and the reviewers’ comments, your manuscript has been accepted for publication in Access Microbiology.
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Comments to Author
All information seems to be there but some parts require a more in depth explanation, and the methods section slightly restructured so that the findings of the paper are clear. L1 - Use the phrase Taqman PCR assays instead of Taqman PCRs, as that is what has been created here L45 - Here it says amplification rates of 100%, 92% and 20% for 1000, 100 and 10 copies respectively for NS3, but in table 2 it says 100%, 100% and 92% for those copy numbers. L54 - Again use the phrase Taqman PCR assays throughout the whole manuscript L69 - Some more background on named viruses such as single or double stranded, enveloped etc L108-117 - This paragraph could be better structured - i.e. starting with where samples were obtained from and how they were handled and/or treated, with machinery used for each step …
Comments to Author
All information seems to be there but some parts require a more in depth explanation, and the methods section slightly restructured so that the findings of the paper are clear. L1 - Use the phrase Taqman PCR assays instead of Taqman PCRs, as that is what has been created here L45 - Here it says amplification rates of 100%, 92% and 20% for 1000, 100 and 10 copies respectively for NS3, but in table 2 it says 100%, 100% and 92% for those copy numbers. L54 - Again use the phrase Taqman PCR assays throughout the whole manuscript L69 - Some more background on named viruses such as single or double stranded, enveloped etc L108-117 - This paragraph could be better structured - i.e. starting with where samples were obtained from and how they were handled and/or treated, with machinery used for each step included, instead of a list of machinery/equipment. L124 - Typo in Thermofisher L131 - Somewhere in the methods section I would suggest including the definition of limit of detection in relation to PCR assays, i.e. lowest concentration of RNA/DNA with 95% positive replicates. Limit of quantification (LOQ) could also be included but is not strictly necessary. LOD and/or LOQ values would be cleared if reported using the copy number - i.e. the LOD for these assays would have to be 100 copies reaction-1 (using the values reported in table 2). Based on those values the true LOD value is likely somewhere between 10 and 100, so a lower LOD value could be achieved if the experiment was run using a narrower range but is not strictly necessary. L167 - As this explains modification of the assay to certain parameters, this needs to be made clearer in the beginning of the manuscript - i.e. why these parameters are used and what advantage that has L173 - this should also be in methods (sequences downloaded from NCBI…) L176 - The LODs for these assays should have a stated definitive value somewhere. L212 - May be worth nothing that many tests on 1 plate is also more cost effective as an additional advantage L253 - If using acronyms I need them to be defined somewhere Figures and tables are clear and communicate their message well
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Thank you for submitting your manuscript for publication in Access Microbiology. It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology. However, based on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication. I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments. Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers’ comments.
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Comments to Author
The manuscript describes the development of two new RT-qPCR assays for the detection of Alongshan Virus (ALSV). Overall, the methods and results appear to be scientifically sound. However, a lot of information and details are missing. The manuscript is difficult to follow and would benefit from a clearer structure. There are also inconsistencies in the used terminology. Please see below for specific comments. Abstract L33: Why is it important to develop and validate new RT-qPCR assays? L36-37: The aim of the manuscript is to develop new RT-qPCR assays for Alongshan Virus (ALSV). While the role of ALSV in meningoencephalitis in humans is not investigated in this manuscript, rapid and precise diagnostics of ALSV will help to further understand the role the virus plays in meningoencephalitis. …
Comments to Author
The manuscript describes the development of two new RT-qPCR assays for the detection of Alongshan Virus (ALSV). Overall, the methods and results appear to be scientifically sound. However, a lot of information and details are missing. The manuscript is difficult to follow and would benefit from a clearer structure. There are also inconsistencies in the used terminology. Please see below for specific comments. Abstract L33: Why is it important to develop and validate new RT-qPCR assays? L36-37: The aim of the manuscript is to develop new RT-qPCR assays for Alongshan Virus (ALSV). While the role of ALSV in meningoencephalitis in humans is not investigated in this manuscript, rapid and precise diagnostics of ALSV will help to further understand the role the virus plays in meningoencephalitis. L38-42: More details in the Methodology section. L40: What technical features? L54: Replace "real-time PCRs" with RT-qPCR assays. L55: Remove "such as ours." L57: Replace "This PCR" with "these assays." Introduction A paragraph describing Alongshan Virus (ALSV) is missing. Please add several sentences including RNA (negative or positive strand) virus, genome size, composition, genetic diversity of the virus, information on the two targets (NS3 and NS5 genes). L71: Abbreviation for TBEV (Tick-borne encephalitis virus) needs to be mentioned in the text. L72-73: Remove "by the same group." L80: Be consistent with abbreviations for TBEV. L100: Remove "this article" and replace with "this study described." L101: ALSV RT-qPCR assay development. Material and Methods Methods are hard to follow. Change the order to follow a similar structure: sample acquisition, handling and nucleic acid extraction, quality control, primer design, assay validation (assay specificity, sensitivity, and efficiency), data analysis. Important information on assay validation is missing. Please see below for specific comments. L108: It seems like RT-PCR and real-time PCR have been used interchangeably throughout the manuscript resulting in misleading use of acronyms. I would advise using RT-qPCR for reverse transcription quantitative real-time PCR. L110: Which kit was used for extraction on MagNA Pure 96? Small viral kit? L114: Hold stage = reverse transcription? L115: Remove "PCR stage." L116: Provide data on annealing temperature optimization. L116: How many replicates per sample? L125: What is the final reaction volume: 20 or 25 µL? Please clarify. L125-126: Why were NS3 and NS5 genes selected for the RT-qPCR assay? Are those conservative regions? L127-128: Data on optimization of primer and probe concentrations is missing. L132: Standard curve is missing from Results. L151-152: Was there an opportunity to sequence the PCR product to confirm assay specificity? L157: It is unclear how one-way ANOVA was used, please clarify. Results L168: LNA = Locked Nucleic Acid? Is increased Tm needed to run the assays in combination with other assays for screening different pathogens? L173-174: Primer design needs to be moved to Methods. Provide more details like how many sequences were downloaded and used, what program was selected for primer design, etc. Any in silico testing of the assays? L177: How many replicates were used to determine the Limits of Detection? Report also efficiency and slope. L186: "Both PCRs" do you mean assays? Change throughout the text. L194: Replace "our region" with the actual location. L195: 71 samples should be mentioned in Methods. L200: What is meant under PCRs here? The newly developed RT-qPCR assays? L200-201: How many samples were tested? L212: Provide information on the automated system in Methods (instead of listing equipment in the RT-PCR platform, instruments, and program). L218: The last sentence needs a reference. L225-226: Was the main goal to develop an assay that has the same annealing temperature as the rest of the pathogen targets tested in the lab? Multiplexing is crucial in clinical settings to generate fast results, however, this needs to be clearly outlined. Was multiplexing an option? L227: 384 plate instead of 348? L228: Give more information on primer design and in silico testing in Methods and Results. L230: "Observed genes target polymorphism" please elaborate and provide more information in Introduction. L231: Not PCRs but assays. L231-233: Re-write the sentence. L231-235: Repeating Results. L241: Have you tried to multiplex NS5 and NS3 assays? L255-256: How does the study highlight the value of automated molecular diagnostic platforms? L268: In Vitro Diagnostic Regulation (IVDR) and the In Vitro Diagnostic Medical Device Ordinance (IVDO). Table 1: Tm should be the annealing temperature that was used, not of each primer separately. Is the length provided in the table the fragment size? Were plasmids ordered as RNA? Table 2: Provide the number of replicates. Figure 2: Please add sample size and graphs with standard curves for each assay.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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