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Development and Evaluation of qPCR Assay for Quantitation of Kazachstania slooffiae and Total Yeasts Occurring in the Porcine Gut

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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.

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Acknowledgments

This study is a part of the project (Grant UR 244/2-1) funded by the German Research Foundation (DFG). The DFG is not responsible for the content of any information represented in the manuscript. The authors would like to thank H. Riese, H. Marx, and A. Orgis for the technical support.

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Authors and Affiliations

  1. Institute of Agricultural and Nutritional Sciences, Group Animal Nutrition, Martin Luther University Halle-Wittenberg, Theodor-Lieser-Straße 11, 06120, Halle (Saale), Germany

    Vladimir Urubschurov, Wolfgang-Bernhard Souffrant & Annette Zeyner

  2. Chair of Nutrition Physiology and Animal Nutrition, Faculty of Agricultural and Environmental Sciences, University of Rostock, Justus-von-Liebig-Weg 8, 18059, Rostock, Germany

    Kirsten Büsing & Wolfgang-Bernhard Souffrant

  3. Karl-Heinrich-Ulrichs-Str. 8a, 10787, Berlin, Germany

    Pawel Janczyk

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  1. Vladimir Urubschurov
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  2. Kirsten Büsing
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Correspondence to Vladimir Urubschurov.

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Urubschurov, V., Büsing, K., Janczyk, P. et al. Development and Evaluation of qPCR Assay for Quantitation of Kazachstania slooffiae and Total Yeasts Occurring in the Porcine Gut. Curr Microbiol 71, 373–381 (2015). https://doi.org/10.1007/s00284-015-0862-2

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