One-Step PCR Detection of Salmonella Pullorum/Gallinarum Using a Novel Target: The Flagellar Biosynthesis Gene flhB

Abstract

Salmonella enterica serovar Pullorum/Gallinarum is an important infectious pathogen that has caused widespread problems for chicken industry. Traditional Salmonella serotyping is an expensive and time-consuming process. In this study, we developed a rapid one-step polymerase chain reaction (PCR) method to identify S. Pullorum/Gallinarum. The PCR-based assay focuses on flhB, which shows a deficient region only in S. Pullorum/Gallinarum, compared with that of other serovars. The specificity and sensitivity of the PCR system were evaluated. The developed PCR method could identify S. Pullorum/Gallinarum from 27 different Salmonella serovars and eight non-Salmonella pathogens. The minimum limit of DNA and the lowest number of cells of S. Pullorum for the PCR detection were no less than 5.85 pg/μL and 10 CFU, respectively. The method was applied to the analysis of Salmonella strains isolated from the chicken farm. The PCR-based testing results of the farm isolates were in concordance with those obtained using traditional serotyping method. This newly developed PCR-based system could be used to accurately screen for the presence of S. Pullorum/Gallinarum, and support traditional serotyping methods, especially in high-throughput screening situations.

Keywords: PCR detection; Salmonella Pullorum/Gallinarum; chicken farm; flhB; one-step.

FIGURE 1

Schematic for the primer design of flhB to distinguish Salmonella Pullorum/Gallinarum from other serovars. flhB gene of S. Pullorum/Gallinarum has a deficient region compared with that of other serovars, which was exploited to design the primers. The red arrows indicate the positions of the designed primers. The PCR amplifies a product of 182 bp of S. Pullorum/Gallinarum and 379 bp of non-S. Pullorum/Gallinarum. SP/SG was referred to S. Pullorum/Gallinarum.

FIGURE 2

Specificity of the one-step PCR for the detection of Salmonella Pullorum/Gallinarum. The single PCR assays, using genomic DNA from various Salmonella and non-Salmonella strains, were conducted using the designed primers targeting flhB. The PCR amplifies a product of 182 bp of S. Pullorum/Gallinarum. Lane M: DL2000 DNA marker (Takara Biotechnology Co., Dalian, China). Detailed strain information is given in Table 1.

FIGURE 3

Sensitivity of the one-step PCR assay for detection of genomic DNA and cells from Salmonella Pullorum (strain S06004). The PCR amplifies a product of 182 bp. Lane M: DL2000 DNA marker (Takara Biotechnology Co., Dalian, China). (A) The PCR for the detection of genomic DNA, lanes 1–8, S. Pullorum genomic DNA used as template at the following concentrations, respectively: 58.5 ng/μL, 5.85 ng/μL, 585 pg/μL, 58.5 pg/μL, 5.85 pg/μL, 585 fg/μL, 58.5 fg/μL, 5.85 fg/μL; (B) The PCR for the detection of S. Pullorum cells, lanes 1 to 6, the number of cells per PCR assay, respectively: 10⁴ CFU, 10³ CFU, 10² CFU, 10¹ CFU, 10⁰ and 10^-1 CFU.

FIGURE 4

One-step PCR for the detection of Salmonella Pullorum/Gallinarum from Salmonella isolates from one chicken farm. The PCR assay produces a target amplicon of 182 bp of S. Pullorum/Gallinarum. Lane M: DL2000 DNA marker (Takara Biotechnology Co., Dalian, China). SP/SG was referred to S. Pullorum/Gallinarum. Detailed information on the Salmonella isolates is given in Table 2.