Development of a multiplex PCR assay for the simultaneous and rapid detection of six pathogenic bacteria in poultry

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.

Keywords: Avian; Multiplex PCR assay; Six pathogenic bacteria; Specificity.

Fig. 1

Amplification of target gene of the multiplex PCR. Lane M: 2000 bp DNA marker; Lanes 1–6: the template of m-PCR respectively were Escherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus; Lane 7: negative control

Fig. 2

Optimization of the primers of m-PCR. Lane M: 2000 bp DNA marker; Lane 1: double PCR formed by Proteus mirabilis and Salmonella spp.; Lane 2: triple PCR formed by Proteus mirabilis, Salmonella spp. and Pasteurella multocida; Lane 3: quadruple PCR formed by Proteus mirabilis, Salmonella spp., Pasteurella multocida and Pseudomonas aeruginosa; Lane 4: quintuple PCR formed by Proteus mirabilis, Salmonella spp., Pasteurella multocida, Pseudomonas aeruginosa and Escherichia coli; Lane 5: sextuple PCR formed by Proteus mirabilis, Salmonella spp., Pasteurella multocida, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus

Fig. 3

Determination of time of the multiplex PCR. Lane M: 2000 bp DNA marker; Lane 1: 24 running cycles; Lane 2: 25 running cycles; Lane 3: 26 running cycles; Lane 4: 27 running cycles; Lane 5: 28 running cycles; Lane 6: 29 running cycles; Lane 7: 30 running cycles; Lane 8: 31 running cycles

Fig. 4

Determination of specificity of the multiplex PCR. Lane M: 2000 bp DNA marker; a Lane 1: the template of m-PCR contain 6 bacterial genomes as positive control. Lane 2: the template of m-PCR was Klebsiella pneumoniae; Lane 3: the template of m-PCR was Shigella spp.; Lane 4: the template of m-PCR was Bacillus subtilis; Lane 5: the template of m-PCR was Bacillus cereus; Lane 6: the template of m-PCR was Enterococcus faecalis; Lane 7: the template of m-PCR was Listeria monocytogenes; Lane 8: the template of m-PCR was Streptococcus suis; Lane 9: the template of m-PCR was Cryptosporidium baileyi; Lane 10: the template of m-PCR was Eimeria tenella; Lane 11: the template of m-PCR was Newcastle disease virus (NDV); Lane 12: the template of m-PCR was Avian Influenza virus H9N2; Lane 13: the template of m-PCR was infectious bursal disease virus (IBDV); Lane 14: Negative control. b Lanes 1–3: the template of m-PCR was O₁, O₂ and O₇₈ serotype of avian pathogenic Escherichia coli, respectively; Lanes 5–7: the template of m-PCR was Salmonella typhimurium, Salmonella enteritidis, Salmonella pullorum, respectively; Lanes 4, 8: negative control

Fig. 5

Determination of the sensitivity of the multiplex PCR for bacterial genomic DNA detection. Lane M: 2000 bp DNA marker; a Lanes 1–10: the concentration of Escherichia coli DNA were 100 ng, 75 ng, 50 ng, 25 ng, 12.5 ng, 7.5 ng, 2.5 ng, 1 ng, 500 pg and 250 pg, respectively. b Lanes 1–10: the concentration of Pasteurella multocida DNA were 100 ng, 75 ng, 50 ng, 25 ng, 12.5 ng, 7.5 ng, 2.5 ng, 1 ng, 500 pg and 250 pg, respectively. c Lanes 1-8: the concentration of Proteus mirabilis DNA were 100 ng, 75 ng, 50 ng, 25 ng, 12.5 ng, 7.5 ng, 2.5 ng, 1 ng, 500 pg and 250 pg, respectively. d Lanes 1–10: the concentration of Pseudomonas aeruginosa DNA were 100 ng, 75 ng, 50 ng, 25 ng, 12.5 ng, 7.5 ng, 2.5 ng, 1 ng, 500 pg and 250 pg, respectively. e Lanes 1–10: the concentration of Salmonella spp. DNA were 100 ng, 75 ng, 50 ng, 25 ng, 12.5 ng, 7.5 ng, 2.5 ng, 1 ng, 500 pg and 250 pg, respectively. f Lanes 1–10: the concentration of Staphylococcus aureus DNA concentration were 100 ng, 75 ng, 50 ng, 25 ng, 12.5 ng, 7.5 ng, 2.5 ng, 1 ng, 500 pg and 250 pg, respectively

Fig. 6

Determination of the sensitivity of the m-PCR for bacterial CFU detection. Lane M: 2000 bp DNA Marker. a Lanes 1–4: the template of Escherichia coli respectively were 5 × 10⁵, 5 × 10⁴, 5 × 10³ and 5 × 10² CFU, respectively. b Lanes 1–5: the template of Pasteurella multocida respectively were 6 × 10⁶, 6 × 10⁵, 6 × 10⁴, 6 × 10³ and 6 × 10² CFU, respectively. c Lanes 1–5: the template of Proteus mirabilis respectively were 2.8 × 10⁶, 2.8 × 10⁵, 2.8 × 10⁴, 2.8 × 10³ and 2.8 × 10² CFU, respectively. d Lanes 1–5: the template of Pseudomonas aeruginosa respectively were 8.6 × 10⁶, 8.6 × 10⁵, 8.6 × 10⁴, 8.6 × 10³, 8.6 × 10² CFU, respectively. e Lanes 1–6: the template of Salmonella spp. respectively were 3.2 × 10⁷, 3.2 × 10⁶, 3.2 × 10⁵, 3.2 × 10⁴, 3.2 × 10³ and 3.2 × 10² CFU, respectively. f Lanes 1–5: the template of Staphylococcus aureus respectively were 5.6 × 10⁶, 5.6 × 10⁵, 5.6 × 10⁴, 5.6 × 10³ and 5.6 × 10² CFU, respectively