title: Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks.
abstract: Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10(3) colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Hu
authors.forename: Jing
authors.initials: J
authors.lastname: Tu
authors.forename: Xiangan
authors.initials: X
authors.lastname: Han
authors.forename: Yinyu
authors.initials: Y
authors.lastname: Zhu
authors.forename: Chan
authors.initials: C
authors.lastname: Ding
authors.forename: Shengqing
authors.initials: S
authors.lastname: Yu
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001715:Bird Diseases / Q000382:microbiology*; D015169:Colony Count, Microbial; D053488:DnaB Helicases / Q000235:genetics; D004372:Ducks / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary*; D029968:Escherichia coli Proteins / Q000235:genetics; D045826:Flavobacteriaceae Infections / Q000382:microbiology / Q000662:veterinary*; D005819:Genetic Markers; D006520:Hepatitis, Animal / Q000382:microbiology; D057230:Limit of Detection; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010493:Pericarditis / Q000382:microbiology / Q000662:veterinary; D010538:Peritonitis / Q000382:microbiology / Q000662:veterinary; D060494:Riemerella / Q000235:genetics / Q000254:growth & development; D012481:Salmonella Infections, Animal / Q000382:microbiology*; D019779:Salmonella enterica / Q000235:genetics / Q000254:growth & development
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005819:Genetic Markers; C075518:invA protein, Bacteria; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli; D053488:DnaB Helicases