Real-time PCR method with statistical analysis to compare the potential of DNA isolation methods to remove PCR inhibitors from samples for diagnostic PCR.
A real-time PCR method for fast comparison of different DNA isolation methods to remove PCR inhibitors from samples is presented. A fixed amount of target-200 copies of a 79-bp region of the COCH gene of the zebrafish (Danio rerio)-was added to each PCR reaction together with isolated DNA from different types of samples including chicken feces. Four commercial DNA isolation kits and a chelex-based technique were compared using this method. The copy numbers calculated and the endpoint fluorescence were statistically compared to the values of 22 control samples containing the control target and water instead of isolated DNA, processed together in the same PCR run. The level of the endpoint fluorescence was more often negatively influenced by inhibitors than the copy number calculated, suggesting a more pronounced effect on the plateau phase of the reaction by limiting one or more compounds in the PCR reaction.
Flekna
Gabriele
G
Department of Veterinary Public Health and Food Science, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria. gabiela.flekna@vu-wien.ac.at
Schneeweiss
Wilfried
W
Smulders
Frans J M
FJ
Wagner
Martin
M
Hein
Ingeborg
I
D000818:Animals; D002645:Chickens; D004247:DNA / Q000302:isolation & purification*; D003627:Data Interpretation, Statistical; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015027:Zebrafish
D011933:Reagent Kits, Diagnostic; D004247:DNA