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Klemen Zupancic1, Misa Korva2, Tatjana Avsic Zupanc2, Urska Cepin3, Manca Pirc3, Laura Simdon4 Matjaz Hren 1,3,*, 1 SCINOTE, LLC, USA 2 Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Slovenia 3 BioSistemika, d.o.o., Slovenia 4 Gilson, Inc., USA |
Abstract
Automation has significantly improved human diagnostics in the developed world by increasing patient throughput and decreasing diagnostic variability caused by human interaction. The outbreak and expansion of Zika virus (ZIKV) has increased the need for rapid and reliable diagnostics in the early stages of infection and for virus quantification in clinical studies. Real time PCR (qPCR) has been found to be the most sensitive, specific and rapid detection system for ZIKV detection.
Our study addressed automation of qPCR plate setup for ZIKV detection and quantification which includes management of sam- ples, preparation of sample dilutions, preparation of master mix and their application to the qPCR plate. This was compared to manual qPCR setup which was performed by an experienced lab diagnostician.
In this study samples were tested from five human patients with suspicion on ZIKV infection that were sent for routine diagnostics of ZIKV to the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Slovenia (IMI). RNA was iso- lated from different tissues or fluids including fetus brain, blood, semen, plasma, or urine. Additionally, for an absolute quantifica- tion study, six different ZIKV strains maintained in cell culture lines were analysed. Necessary controls were included in every step of the process. The same samples were used for manual and auto- mated qPCR experiments on the same day.
The automated setup included management of samples in sciNote Open Source Electronic Lab Notebook while sample dilu- tions, master mix preparation and qPCR plate setup was done by using Gilson qPCR Assistant with PIPETMAX® 268 automated pipet- ting workstation (PIPETMAX®).
Analysis of the qPCR data revealed that the accuracy of perfor- mance of automated qPCR plate setup was comparable to manual setup done by a very experienced lab diagnostician while the speed of the setup was improved by automating the sample import, sam- ple dilutions and qPCR plate setup.
Incorporating an automation platform into the diagnostic pro- tocol allows for accurate standard dilution and assay setup with a significant increase in throughput compared to manual processing. Moreover, integration of data management software with an automation system further increases the throughput and reduces the possibility of user error. More importantly, data management software, such as sciNote, enables full traceability of samples and results which is critical for accuracy in human diagnostics.
http://dx.doi.org/10.1016/j.bdq.2017.02.069
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