Detection of Lawsonia intracellularis by one tube nested-PCR

Swine proliferative enteropathy (PE) is caused by an obligate intracellular bacterium Lawsonia intracellularis (L. intracellularis) PE is a serious enteric disease of weaned pigs resulting in growth rate losses, increasing number of death cases and costs of veterinary treatment. The aim of this study was, to develop and evaluate one tube nested - PCR technique for simple and fast L. intracellularis DNA detection in feces. Total DNA was extracted from feces which were taken directly from rectum of weaned piglets and fatteners with diarrhea using the Genomic DNA Prep Plus(R) kit (Helicionus, A&A Biotechnology). Five micro liters of DNA were amplified by two methods (standard and modified in closed one tube). For each method were used two sets of PCR primers, which consisted of 20 - base pair (bp) oligonucleotides corresponding to 319 bp and 570 bp regions of p78 L. intracellularis sequence. The standard method consisting of 2 steps was performed in two separate reaction tubes: first step PCR and second step nested - PCR. In single - tube method both steps were done in one closed tube. In this method all used reagents for first step PCR were deposited in tube bottom, while reagents for nested - PCR were immobilized in a tube cap by carbohydrate trehalose. After first step PCR was completed the tube was vortexed, centrifuged and the nested - PCR was performed. This procedure was evaluated for examination 491 positive and negative fecal samples taken from different pig farms. Both techniques gave the same results wit positive and negative samples. It was concluded that the closed, one tube nested - PCR method was ver sensitive and less prone to give false positive results, compared to standard nested - PCR, carried out in separate reaction tubes. The investigation showed that modified closed one tube nested - PCR method is a useful tool for rapid diagnosis of PE in pig population and could be performed for monitoring of L. intracellularis infections.