title: Development and Evaluation of qPCR Assay for Quantitation of Kazachstania slooffiae and Total Yeasts Occurring in the Porcine Gut.
abstract: Kazachstania slooffiae is the dominating yeast in pig's gut. No methods others than cultivation were applied for enumeration of yeasts within this ecosystem. Therefore, the aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay to quantitate total yeasts and K. slooffiae in the porcine gut. This work demonstrated that the copy numbers in gDNA can be determined by qPCR using PCR amplicons as a calibrator and one-point calibration method. The gDNA were then used as a calibrator for further analysis. The values of quantitation cycle and PCR amplification efficiency of gDNA calibrator were highly reproducible. DNA was extracted from feces and from 10 different cultured yeasts found in pigs' intestine. The qPCR results using primers NL1/LS2 encoding 26S rDNA correlated (r = 0.984, P < 0.0001) with cultivation results. From two primer sets developed, one set encoding act1 gene was suitable for quantitation of K. slooffiae. The copy numbers of K. slooffiae could be determined by 40% analyzed animals, amounting to about 70% of total yeasts. The application of this method in next studies will help to get more information about K. slooffiae and total yeasts in the gut of pigs.
authors.affiliation: Institute of Agricultural and Nutritional Sciences, Group Animal Nutrition, Martin Luther University Halle-Wittenberg, Theodor-Lieser-Straße 11, 06120, Halle (Saale), Germany, urubschurov@web.de.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Urubschurov
authors.forename: Kirsten
authors.initials: K
authors.lastname: Büsing
authors.forename: Pawel
authors.initials: P
authors.lastname: Janczyk
authors.forename: Wolfgang-Bernhard
authors.initials: WB
authors.lastname: Souffrant
authors.forename: Annette
authors.initials: A
authors.lastname: Zeyner
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D041981:Gastrointestinal Tract / Q000382:microbiology*; D012335:RNA, Ribosomal / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D004718:Saccharomycetales / Q000145:classification* / Q000302:isolation & purification*; D013552:Swine; D015003:Yeasts / Q000145:classification* / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S