A Duplex PCR Assay for Rapid Detection of Klebsiella pneumoniae and Chryseobacterium in Large Yellow Croaker Fish

Abstract

Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 μM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.

Keywords: Chryseobacterium; Duplex PCR method; Klebsiella pneumoniae; Sensitivity; Specific.

FIG. 1.

The results of duplex PCR optimization for Klebsiella pneumoniae and Chryseobacterium. M:1kb DNA Marker I; lanes1-4: Duplex PCR using 1, 0.5, 0.25 and 0.125 μM primers (KP-ompA-F/KP-ompA-R for Klebsiella pneumoniae, ompA-F/ompA-R for Chryseobacterium); lane 5: negative control.

FIG. 2.

Specificity of duplex PCR using primers specific for Klebsiella pneumoniae and Chryseobacterium. M: Q8000 DNA Marker. Lanes 1–8: template genomic DNA extracted from Klebsiella pneumoniae, Chryseobacterium, or both Klebsiella pneumoniae and Chryseobacterium, Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila, Acinetobacter johnsonii; lane 9: negative control.

FIG. 3.

Detection limit of the duplex PCR for Klebsiella pneumoniae and Chryseobacterium using genomic DNA (a), Klebsiella pneumoniae (b) and Chryseobacterium (c). Lane M: 1 kb DNA Marker I. a. The template was genomic DNA extracted from Klebsiella pneumoniae and Chryseobacterium and subjected to 10-fold serial dilution. Lanes 1–8: The amounts of the DNA template were 20 ng, 2 ng, 200 pg, 20 pg, 2 pg, 200 fg, 20 fg, and 2 fg. line 9: negative control (without template); b and c: lanes 1–5: serial tenfold dilutions of the chromosomal DNA extracted from the indicated bacteria, 10⁵ (lane 1) to 10 (lane 5) CFU.

FIG. 4.

Duplex-PCR reproducibility assay using genomic DNA isolated from the target strains at different cell concentrations (a) and three replicates of each concentration (b). M:1 kb DNA Marker I; a. Lanes 1–3: The template genomic DNA extracted from Klebsiella pneumoniae and Chryseobacterium at concentrations of 10⁶, 10⁴ and 10³ CFU/mL, respectively. N: negative control (without template); b. Lanes 1–3: three replicates at the concentration of 10³ CFU/mL. Lanes 4–6: three replicates of 10⁴ CFU/mL. Lanes 7–9: three replicates of 10⁶ CFU/mL.

FIG. 5.

Agarose gel electrophoresis of the duplex PCR products obtained using the genomic DNA of each bacterium or both together, mixed with liver tissue from zebrafish. Lane 1, mixture of genomic DNA from the Klebsiella pneumoniae and liver tissue; lane 2, mixture of genomic DNA from the Chryseobacterium and liver tissue; lane 3, mixture of genomic DNA of both Chryseobacterium and Klebsiella pneumoniae, combined with liver tissue; line 4: negative control (without template); M: 1 kb DNA Marker I.

FIG. 6.

Testing of clinical samples using duplex PCR. M: 1 kb DNA Marker I. Lines 1–20: the genomic DNA was extracted from clinical samples comprising liver tissue of diseased fish.