Molecular Identification of Oxalobacter formigenes with Polymerase Chain Reaction: Fresh vs. Frozen Stool

Kwak, Cheol; Jung, Byong Chang; Kim, Hee Kyung; Lee, Jung Hee; Kim, Eui Chong; Kim, Hyeon Hoe

Abstract

PURPOSE: Oxalobacter formigenes is an intestinal flora degrading oxalate in the gut. However, microbiological detection of this organism is quite difficult. We tried to develop a simple, rapid and cost-effective PCR method for detecting Oxalobacter formigenes from fecal specimens and to determine whether O. formigenes could be detected from frozen fecal specimens as well as fresh stool.
MATERIALS AND METHODS
Whole bacterial DNA was isolated directly from fresh and frozen stool samples obtained from 30 healthy volunteers known to be free from urolithiasis and from fresh stool samples obtained from 38 patients with urolithiasis. Genus specific oligonucleotide sequences corresponding to homologous regions residing in the oxc gene that encodes for oxalyl-coenzyme A decarboxylase were designed. A PCR-based assay was done in both fresh and frozen stool samples and the nucleotide sequences were analyzed to determine the details of oxc.
RESULTS
PCR product of 416-bp molecular size encoding oxc gene was detected in 23 (77%) of 30 healthy volunteers and in 14 (37%) of 38 patients with urolithiasis. In healthy volunteers, the results of PCR for the fresh and the frozen stool proved identical in each subject. The nucleotide sequence analysis revealed that the sequence of the amplified product was compatible with that of oxc gene.
CONCLUSIONS
O. formigenes could be identified easily and efficiently by this PCR-based detection system. Furthermore, as the PCR-based assay results in the frozen fecal samples were identical as that of fresh stool, immediate processing of the fecal samples may not be necessary to detect O. formigenes in the clinical setting.