Comparative evaluation of automated KingFisher Flex Purification System 96 (ThermoFisher Scientific) and manual QIAamp Viral RNA Mini Kit (Qiagen) extraction methods for SARS-CoV-2

Abstract

Background: The extraction step of the viral material of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) influences the quality of reverse transcriptase-polymerase chain reaction (RT-PCR) results in diagnosis of coronavirus disease 2019 (COVID-19). The purpose of this cross-sectional study was to evaluate the diagnostic performance of the automated extraction system "KingFisher Flex Purification System 96 (ThermoFisher)" compared to the manual method with the "QIAamp Viral RNA Mini Kit (Qiagen)".

Methodology: From October to December 2020, comparative diagnostic evaluation of two methods of SARSCoV-2 RNA extraction methods was conducted on 159 fresh and 120 frozen nasopharyngeal and oropharyngeal specimens collected from travellers and suspected cases or contacts of COVID-19 patients in Burkina Faso. The FastPlexTM Triplex 1-Step COVID 19 Detection Kit (RT-PCR, RNA extraction free) (Precigenome LLC) was used to amplify on the same PCR plate, RNA extracts from manual QIAamp Viral RNA Mini Kit and automated KingFisher Flex Purification System 96 (ThermoFisher) using the QuantStudio5 thermal cycler (Applied Biosystems). Analysis of the diagnostic performance of the SARS-CoV-2 RT-PCR assay following RNA extraction by the two methods was done using an online OpenEpi software.

Results: For fresh samples, the study found a slightly higher RT-PCR positivity rate following manual extraction (12.6%) than automated extraction (9.4%). For frozen samples, the positivity rate was far higher for manual (38.33%) than automated extraction method (20.83%). The results show that the performance of the automated extraction was inferior when compared to the manual extraction for both fresh samples (sensitivity 35%, specificity 94.2%) and frozen samples (sensitivity 43.5%, specificity 93.2%). However, using McNemar Chi-square with Yates correction, there was no significant difference in positivity rate of RT-PCR (x2=0.76, p=0.38) between the two extraction methods for the fresh samples, but there was a significant difference (x2=12.9, p= 0.0003) in the extraction of the frozen samples.

Conclusion: The results of this study showed that KingFisher Flex Purification System 96 (ThermoFisher) automatic extraction method was less sensitive and specific than QIAamp Viral RNA Mini Kit (Qiagen) manual extraction method. This information can serve as guide to laboratories in the choice of RNA extraction methods to use for RT-PCR detection of SARS-CoV-2.