Development Of A Multiplex-PCR For The Detection Of Lawsonia Intracellularis,Brachyspira Hyodysenteriae,and Clostridium Difficile In Pig Faeces

Lawsonia intracellularis and Brachyspira hyodysenteriae are two common intestinal pathogens of pigs which are widespread in the world and cause huge economic losses to the pig industry.Clostridium difficile is one of the pathogens of zoonosis.In rencent years,the prevalence of pathogenic Clostridium difficile found in pig farms is increasing just as the threat of Clostridium difficile to food safety.In order to improve the economic benefits of the pig industry,ensure food safety and human health,these three pathogens should be widely valued and studied.Swine stool samples are easier to obtain than blood and tissue samples of pigs.They do not cause stress response to pigs.They can be sampled in vivo and are more suitable for the detection of intestinal pathogens.Lawsonia intracellularis,Brachyspira hyodysenteriae and Clostridium difficile can be excreted in the feces,detection of bacterial nucleic acids from feces can be used to confirm whether pigs are infected by these three pathogens.The research objective of this project is to establish a multiplex PCR detection method for simultaneous detection of Lawsonia intracellularis,Brachyspira hyodysenteriae and Clostridium difficile in swine stool.In this study,the cultivation method of Lawsonia intracellularis was explored and Lawsonia intracellularis was successfully cultivated to the fourth generation.The rejuvenation of Brachyspira hyodysenteriae and Clostridium difficile was completed,and the three pathogenic genome were extracted.The large amount of genome is used to establish multiple PCR detection methods for Lawsonia intracellularis,Brachyspira hyodysenteriae and Clostridium difficile.In this study,a complete 16 S rRNA sequence of Lawsonia intracellularis was cloned from pig feces,which proved the existence of Lawsonia intracellularis infection at the pigs farms,and showed that stool samples can be used to verify multiplex PCR detection method for Lawsonia intracellularis Brachyspira hyodysenteriae and Clostridium difficile.The establishment of a multiplex PCR detection method for Lawsonella intracellularis,Brachyspira hyodysenteriae and Clostridium difficile is as follows: First,select Lawsonella intracellular spartate deaminase as the target gene of Lawsonella.Using bioinformatics technology,conservative and specific gene fragments in the genomes of Brachyspira hyodysenteriae and Clostridium difficile were screened as target genes.Second,specific primers were design for the gene of interest.Then,optimization and adjustment of the various components of the reaction system were perform to achieve the best detection effect.The specificity test of this method showed that only specific bands were generated for Lawsonia intracellularis,Brachyspira hyodysenteriae and Clostridium difficile,the minimum detection limits of this method were Lawsonia intracellular DNA 2.6 pg/μL,Brachyspira hyodysenteriae 4 pg/μL,and Clostridium difficile 1.2pg/μL.In totalo,this work was divided into three parts:1.Cultivation and identification of Lawsonia intracellular.2.This study established a multiple PCR method for simultaneous detection of Lawsonia intracellularis,Brachyspira hyodysenteriae and Clostridium difficile in swine feces.The method has the advantages of simple operation,time and labor saving,and low cost.It is applicable to the diagnosis ofLawsonella intracellulaisr,Brachyspira hyodysenteriae and Clostridium difficile infections in pig farms,monitoring and epidemiological investigations of Lawsonia intracellular,Brachyspira hyodysenteriae and Clostridium difficile infections,and further research and prevention References are provided for related pig diseases caused by intracellular Lawsonella,Brachyspira hyodysenteriae and Clostridium difficile.3.Cloning and analysis of 16 S rRNA sequence of Lawsonia intracellularis in swine stool samples.