Methanobrevibacter smithii isolation from human stool using specific rpoB gene

Archaea are Extrtermophile microorganisms and for several decades it has been believed that they are only found in harsh environments, such as volcanoes, deep oceans and salt lakes. However, at present time, their existence in human and mammal’s intestine has been proved. The most important Archaea in human intestine is Methanobrevibacter smithii, which has a major role is some gastrointestinal disorders, as well as obesity. Therefore, Methanogens isolation and detection has such a crucial clinical importance. In this study, we isolated this microorganism for the first time using local technique.
In this study, Archaea DNA was extracted from healthy subject’s stool samples, considering the specific criteria for choosing the healthy group. PCR reaction was performed to amplify the rpoB. Enzyme digestion was operated using restriction enzyme to confirm the rpoB gene. The PCR product was then cloned in E.coli (DH5α) host and sequencing process was performed.
Of 20 stool samples, the rpoB gene was confirmed in 18 samples (90%) and also the AVAII enzyme digestion results proved the gene identity. Sequencing results in NCBI site proved that isolated microorganisms were Methanobrevibacter smithii.
This study revealed that by considering the microorganisms’ variety in intestine, the precise gene detection methods for selecting the specific microbiota, in order to prevent existing similarities between homolog microbiota is vital in microbiota isolation.