Virulence Plasmid (pYV)-Associated Expression of Phenotypic Virulent Determinants in Pathogenic Yersinia Species: A Convenient Method for Monitoring the Presence of pYV under Culture Conditions and Its Application for Isolation/Detection of Yersinia pestis in Food

Figure 4

Confirmation of presence of pYV of Y. pestis in the original strain before subculturing, and CR-positive clones from CR-MOX, CR-BHO, and BHI broth using PCR assay targeting a key regulatory gene virF from pYV. The primer pairs (5′-TCATGGCAGAACAGCAGTCAG-3′ and 5′- ACTCATCTTACCATTAAGAAG-3′) for detection of the virF gene (430- to 1020-nucleotide region) amplified a 591-bp-product from the virulence plasmid. The Lcr-CR⁺ clones showed the presence of 591 bp products from pYV (lanes 2–10 and 12). Lane 1, 50–1,000 bp ladder marker; lane 2, negative control with no template; lane 3, original KIM5 strain as positive control; lanes 4, 5, Lcr-CR⁺ colonies from the CR-MOX and CR-BHO respectively (Figure 5; no. 1); lane 6 BHI broth (Figure 5; 1st passage; no. 2); lane 7 stock culture on CR-MOX (no. 3; 1st passage); lane 8 stock culture on CR-BHO (no. 3; 1st passage); lane 9 BHI broth (no. 4, 2nd passage from CR-MOX); lane BHI broth 10 (no. 4, 2nd passage from CR-BHO); lane 11 (no. 5, 2nd passage on CR-MOX) showing the absence of 591-bp product, and lane 12 (no. 5, 2nd passage on CR-BHO) [30].