Inhibitor-free DNA for real-time PCR analysis of synovial fluid from horses, cattle and pigs.
The potential of five different commercial DNA isolation methods to remove real-time PCR inhibitors from the synovial fluid of horses, cattle and pigs was investigated. All kits with the exception of one included a silica column-based purification of the DNA. With the fifth kit, DNA purification is achieved by removing contaminating macromolecules by a desalting process. We used a recently developed method based on comparison of the real-time PCR signal of an artificial target incorporated into each PCR reaction in the presence of the isolated DNA from the sample, and in control samples containing water instead of isolated DNA. This was followed by statistical analysis of the data. Inhibition and subsequent reduction of the endpoint fluorescence in the real-time PCR reaction was encountered in many cases. Less frequently, the target copy number in the samples was underestimated. However, we found no experimental evidence of a negative influence of the reduced endpoint fluorescence signal on the detection limit of the real-time PCR assay. All kits tested were useful for analyzing pelleted synovial fluid from horses, cattle and pigs. When analyzing non-pelleted synovial fluid, three kits - two based on silica columns and one employing a desalting process - yielded inhibitor-free DNA for real-time PCR analysis.
Schneeweiss
Wilfried
W
Clinic for Orthopaedics in Ungulates, Department for Small Animals and Horses, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.
Stanek
Christian
C
Wagner
Martin
M
Hein
Ingeborg
I
D000820:Animal Diseases / Q000175:diagnosis / Q000382:microbiology*; D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D006734:Horse Diseases / Q000175:diagnosis / Q000382:microbiology; D006736:Horses; D007592:Joint Diseases / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000382:microbiology; D013582:Synovial Fluid / Q000737:chemistry / Q000382:microbiology*
D004269:DNA, Bacterial