Development of a Terminal Restriction Fragment Length Polymorphism analysis protocol for comparing bacterial populations in the porcine gut

Extract

Disease in a pig herd can have major economic impacts, hampering agricultural processes and creating barriers to trade. Importantly, an outbreak of disease can also pose a risk to human health. It is currently unknown what effects different rearing regimes might have on the incidences of zoonoses in pigs. Outdoor rearing of pigs has gained popularity recently due to interest in animal welfare and an increase in the marketability of organic food. But it is unknown if outdoor rearing can alter the gut microbiology of pigs, and if pigs reared outdoors are more susceptible to zoonotic infections. A method for analysing bacterial populations present in the pig gut has been developed based on amplification of the 16S ribosomal DNA. This technique, known as Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis has been used for studying bacterial populations in environments such as soil (Osborn et al., 2000) and faeces (Li et al., 2007). It uses fluorescently labelled forward and reverse primers to generate labelled amplicons, followed by a restriction endonuclease digest of the amplified DNA to give rise to labelled terminal fragments that vary between different species. These terminal fragments are then detected using electrophoretic separation and laser detection, and identified based on the fragment size. This study aims to develop a protocol for using this technique on pure cultures of control organisms.