Abstract
Escherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus are six bacterial pathogens of avian. However, these pathogens may cause many similar pathological changes, resulting in clinical isolates that are difficult to quickly and simultaneously detect and identify. Here, a multiplex polymerase chain reaction (m-PCR) assay is reported to rapidly identify targets genes (phoA, KMT1, ureR, toxA, invA, and nuc) of these six pathogens in clinical samples. Six pairs of specific primers were designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results showed that betaine remarkably improved amplification of the target genes. Specific test results showed that all six pathogens were detected by the proposed m-PCR protocol without cross-amplification with viruses or parasites. Sensitivity test results showed that the m-PCR system could amplify the six target genes from bacterial genomes or cultures with template amounts of 500 pg or 2.8–8.6 × 103 colony forming units, respectively. Furthermore, the six bacterial pathogens isolated from the infected tissue samples were successfully identified. The proposed m-PCR assay is a useful tool to monitor and diagnose bacterial infection in birds with high specificity, sensitivity and throughput.
Highlights
Several factors have been linked to the spread of pathogenic bacteria to poultry, including the expansion of the poultry industry, the increased mobility of humans and animals, water pollution, environmental climate change (Rodriguez-siek et al 2005; Benskin et al 2009)
Number of colony forming units (CFU) of the six pathogens The plate counting results showed that amounts of Escherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus were 5.00 × 108, 3.08 × 108, 1.41 × 109, 4.28 × 109, 1.88 × 109, and 2.79 × 109 CFU at OD600 of 1.0, respectively
The results indicated that different serotypes of avian pathogenic Escherichia coli (Fig. 4b, lanes 1–3) or Salmonella spp. (Fig. 4b, lanes 5–7) could be detected by multiplex polymerase chain reaction (m-polymerase chain reaction (PCR)) assay
Summary
Several factors have been linked to the spread of pathogenic bacteria to poultry, including the expansion of the poultry industry, the increased mobility of humans and animals, water pollution, environmental climate change (Rodriguez-siek et al 2005; Benskin et al 2009). The failure to diagnose the bacterial diseases of poultry,. A variety of methods have been established for the effective diagnosis of avian bacterial diseases, which include antigen-specific enzyme-linked immunosorbent assays (ELISAs), immunogold labeling and various other molecular biology techniques (Kotetishvili et al 2002; Yano et al 2007; Reischl 1996), especially polymerase chain reaction (PCR) technologies. The failure of multi-pathogen detection still was one of major deficiencies to these detection methods. Park et al (2011) established a triple PCR method for analysis of Campylobacter spp., Escherichia coli O157:H7 and Salmonella serotypes. Hu et al (2011) established a triple PCR method for analysis of Riemerella anatipestifer, Escherichia coli (E. coli) and Salmonella with high Park et al (2011) established a triple PCR method for analysis of Campylobacter spp., Escherichia coli O157:H7 and Salmonella serotypes. Hu et al (2011) established a triple PCR method for analysis of Riemerella anatipestifer, Escherichia coli (E. coli) and Salmonella with high
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