Comparative evaluation of in-house manual, and commercial semi-automated and automated DNA extraction platforms in the sample preparation of human stool specimens for a Salmonella enterica 5'-nuclease assay.
In the present study, three methods (NucliSens miniMAG [bioMérieux], MagNA Pure DNA Isolation Kit III Bacteria/Fungi [Roche], and a silica-guanidiniumthiocyanate {Si-GuSCN-F} procedure for extracting DNA from stool specimens were compared with regard to analytical performance (relative DNA recovery and down stream real-time PCR amplification of Salmonella enterica DNA), stability of the extracted DNA, hands-on time (HOT), total processing time (TPT), and costs. The Si-GuSCN-F procedure showed the highest analytical performance (relative recovery of 99%, S. enterica real-time PCR sensitivity of 91%) at the lowest associated costs per extraction (euro 4.28). However, this method did required the longest HOT (144 min) and subsequent TPT (176 min) when processing 24 extractions. Both miniMAG and MagNA Pure extraction showed similar performances at first (relative recoveries of 57% and 52%, S. enterica real-time PCR sensitivity of 85%). However, when difference in the observed Ct values after real-time PCR were taken into account, MagNA Pure resulted in a significant increase in Ct value compared to both miniMAG and Si-GuSCN-F (with on average +1.26 and +1.43 cycles). With regard to inhibition all methods showed relatively low inhibition rates (< 4%), with miniMAG providing the lowest rate (0.7%). Extracted DNA was stable for at least 1 year for all methods. HOT was lowest for MagNA Pure (60 min) and TPT was shortest for miniMAG (121 min). Costs, finally, were euro 4.28 for Si-GuSCN, euro 6.69 for MagNA Pure and euro 9.57 for miniMAG.
Schuurman
Tim
T
Department of Research and Development, Laboratory for Infectious Diseases, Groningen, The Netherlands.
de Boer
Richard
R
Patty
Rachèl
R
Kooistra-Smid
Mirjam
M
van Zwet
Anton
A
D001681:Biological Assay; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification*; D004720:Endonucleases; D005243:Feces / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic / Q000191:economics; D015203:Reproducibility of Results; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic; D004720:Endonucleases