S.H. Stauffer; A.J. Birkenheuer; M.G. Levy; H. Marr; J.L. Gookin
Feces are increasingly recognized as practical samples for molecular diagnosis of infectious disease. Extraction of PCR-quality DNA from feces can be challenging due to co-extraction of PCR inhibitors. Accordingly, we examined the effect of four commercially-available DNA extraction methods on sensitivity of PCR for detection of Tritrichomonas foetus (TF) in naturally-infected and TF-spiked feline stool.
Kits evaluated included ExtractMaster Fecal DNA Extraction Kit, Epicentre Biotechnologies (Kit A); QIAamp DNA Stool Mini Kit, Qiagen (Kit B); UltraClean Fecal DNA Kit, MoBio (Kit C); and ZR Fecal DNA Kit, Zymo Research (Kit D). In accordance with manufacturer instructions, DNA was extracted from 180mg (A,B), 50mg (C), 100 & 150mg (D) aliquots of feline feces to which was added 20µl volumes containing 0-10,000 cultured feline TF. Each kit was also used to extract DNA from the feces of each of 10 naturally infected and 10 uninfected cats. DNA was eluted in 300 µl (A), 200 µl (B), 50 µl (C), or 100 µl (D) of respective elution buffer. Endogenous PCR inhibitors in extracted DNA was examined by PCR amplification of an 876 bp gene fragment of bacterial 16S rRNA. DNA was then tested by single tube nested PCR for amplification of partial ITS1, 5.8S and ITS2 rRNA genes of TF.
Kit D provided the most sensitive detection of TF DNA as expressed by both organisms per DNA extraction and organisms per PCR reaction. To account for differences in DNA concentrations between kits (i.e., fecal sample size and elution volumes), the limit of detection for each kit as expressed by the number of TF per PCR reaction was as follows; Kit B = 250, Kit A = 167, Kit C = 100, Kit D (150 mg fecal sample) = 5, and Kit D (100 mg fecal sample) = 0.5. PCR performed on DNA extracted from cultured TF (no feces) or TF-spiked feces (100mg) using Kit D was positive with as few as 10 TF per extraction. Further, DNA extraction using Kit D could be completed in the shortest time of all kits tested.
These studies identify the ZR Fecal DNA Kit as superior to the other kits tested for extraction of PCR-quality DNA from feline feces.