An acceptable method to evaluate the analytical performance of real-time fluorescent RT-PCR targeting SARS-CoV-2
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
It was necessary to carry out methodologies evaluations of real-time fluorescent reverse-transcription PCR (RT-PCR) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Considering biosafety issues and lack of positive specimens in some special locations in China, the routine specimens from healthy individuals were used to perform methodologies evaluations, in which the indexes were the differences of quantification cycle values (ΔCq) between human derived internal reference control (IRC) genes of a specimen and quality control (QC). Serial experiments were carried out to evaluate various factors that might affect aforementioned methodologies, such as types of virus transport mediums, methods of specimen pretreatment and template preparation, specimen vortex strength, specimen storage temperature and duration. The results showed that using ΔCq values as indexes, among various factors that might affect analytical performance, it was better to store specimens in the normal saline transport mediums, inactivate pathogens using water or metal bath, release more virus particles from swabs by vortex mixing, extract nucleic acids with centrifuge methods, and perform amplification assays timely. Aforementioned opinions and optimum conditions were further confirmed by SRAS-CoV-2 pseudovirus and clinical positive specimens. Altogether, the results of this study indicated that the routine specimens from healthy individuals could be used to evaluate the analytical performance of real-time fluorescent RT-PCR targeting SRAS-CoV-2, of which the indexes were the ΔCq values between IRC genes of a specimen and QC. This acceptable method was extremely valuable in both theoretical and practical significance under current pandemic of coronavirus disease 2019 (COVID-19).
Article activity feed
-
SciScore for 10.1101/2020.05.18.20105247: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: This study was approved by the ethics committee of Daping Hospital.
Consent: Written informed consent was obtained from healthy volunteers, patients or their family members prior to specimen collection.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:A common limitation to PCR-based …
SciScore for 10.1101/2020.05.18.20105247: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: This study was approved by the ethics committee of Daping Hospital.
Consent: Written informed consent was obtained from healthy volunteers, patients or their family members prior to specimen collection.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:A common limitation to PCR-based method is failed amplification due to the presence of PCR-inhibitory substances in the specimens, such as heme compounds found in blood, aqueous and vitreous humors, heparin, urine, polyamines, plant polysaccharides [20]. Based on the current comparative analysis on various types of virus transport mediums, it was essential to select the types of virus transport mediums when CF or OS method of template preparation was planned to be used in real-time fluorescent RT-PCR targeting certain pathogens. For example, due to the presence of amplification inhibitor, i.e., guanidine salt [21, 22], the analytical performance of NX mediums was always lower than that of both NS and YK mediums (Fig. 2A). Considering biosafety of RT-PCR detections targeting SARS-CoV-2, it was better to inactivate pathogens using water or metal bath before any followed procedures because there were no significant differences between various methods of specimen pretreatment (Fig 1). For the other factors that might affect the analytical performance of SARS-CoV-2 nucleic acid detections, it was better to implement analysis in time (Fig. 3), and release SARS-CoV-2 particles from oropharyngeal swabs by vortex mixing (Fig. 2B).
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
-
