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Use of macroporous polypropylene filter to allow identification of bacteria by PCR in human fecal samples
A Cavallini1, M Notarnicola, P Berloco
1Laboratory of Biochemistry, I.R.C.C.S. S. de Bellis, Scientific Institute for Digestive Diseases, Castellana Grotte (BA), Italy.
Abstract:
The detection of pathogenic bacteria directly in human fecal specimens by PCR, requires removal of PCR-inhibitory substances. To investigate whether five different macroporous filters (polypropylene, nylon, polyester, polyethylene, fluorocarbon) could retain polysaccharides, major PCR inhibitors, an in vitro model and human fecal samples were used. The in vitro model consisted of Xanthum gum solutions (3 mg/ml PBS), a bacterial polysaccharide, to which Helicobacter pylori cells were added. Fecal samples from healthy volunteers were spiked with H. pylori and Mycobacterium paratuberculosis cells. Polysaccharide concentrations were significantly reduced only by the polypropylene but not by the other filters. Accordingly, both Xanthum gum solutions and spiked fecal specimens became PCR positive only after filtration with the polypropylene filter. We conclude that this filter can be used to prepare a bacterial DNA template suitable for PCR analysis from human feces.
Insights
Polypropylene filters effectively remove PCR inhibitors from human fecal samples. This filtration method enables accurate detection of pathogenic bacteria using polymerase chain reaction (PCR).
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Polymerase chain reaction (PCR) is crucial for detecting pathogenic bacteria in human feces.
- PCR assays can be inhibited by substances like polysaccharides present in fecal samples.
- Effective removal of inhibitors is necessary for reliable bacterial detection.
Purpose of the Study:
- To evaluate the efficacy of five macroporous filters (polypropylene, nylon, polyester, polyethylene, fluorocarbon) in retaining polysaccharides.
- To determine if filtration can remove PCR inhibitors from human fecal specimens.
- To identify a suitable filtration method for preparing bacterial DNA templates for PCR analysis.
Main Methods:
- An in vitro model using Xanthum gum solutions (a bacterial polysaccharide) and Helicobacter pylori cells.
- Human fecal samples from healthy volunteers spiked with H. pylori and Mycobacterium paratuberculosis cells.
- Filtration of samples through five different macroporous filter types.
Main Results:
- Only polypropylene filters significantly reduced polysaccharide concentrations.
- Other filters (nylon, polyester, polyethylene, fluorocarbon) did not effectively retain polysaccharides.
- PCR analysis of fecal samples became positive only after filtration with the polypropylene filter.
Conclusions:
- Polypropylene macroporous filters are effective in removing PCR-inhibitory polysaccharides from human fecal samples.
- This filtration method yields a bacterial DNA template suitable for PCR-based detection.
- The findings support the use of polypropylene filters for improved bacterial diagnostics in clinical settings.