Figure 1The molecular size of polymerase chain reaction (PCR) fragments amplified with species-specific primers. Agarose gel electrophoresis showing the results from a PCR amplification of genomic DNA from three Vibrio spp. Lane M: 100 bp DNA ladder; Lane 1, Lane 2, and Lane 3: amplified DNA using oligonucleotide primers specific for the hlyA, vvhA, and tlh genes showing specific bands of DNA of the expected sizes of 57 bp, 79 bp, and 58 bp, respectively.
Figure 2Multiplex real-time polymerase chain reaction (PCR) amplification plots and standard curves of single target genes for Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, respectively. Standard curves were plotted for the log cell number of bacteria versus the number of cycles required to reach Ct. Samples were derived from DNA extracted from 10-fold dilutions of cells at concentrations of 101 – 107 colony-forming units/reaction. Although all samples were assayed in duplicate, only a single replicate is displayed for each sample for clarity. The equations of the lines obtained for V. cholerae, V. parahaemolyticus, and V. vulnificus were y = –4.767x + 49.113 (R2 = 0.9831), y = –3.798x + 44.416 (R2 = 0.9915), and y = –3.678x + 43.741 (R2 = 0.9966), respectively.
Figure 3Multiplex real-time polymerase chain reaction (PCR) amplification plots and standard curves of three target genes for Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, respectively. Standard curves were plotted for the log cell number of bacteria versus the number of cycles required to reach Ct. Samples were derived from DNA extracted from 10-fold dilutions of cells at concentrations of 101 – 107 colony-forming units/reaction. Although all samples were assayed in duplicate, only a single replicate is displayed for each sample for clarity. The equations of the lines obtained for V. cholerae, V. parahaemolyticus, and V. vulnificus were y = –4.883x + 50.954 (R2 = 0.9816), y = –4.286x + 48.783 (R2 = 0.9995), and y = –4.093x + 46.869 (R2 = 0.9988), respectively.
Table 1Strains tested for assay specificity
Species (No. isolates) |
Gene target
|
hlyA
|
tlh
|
vvhA
|
Vibrio cholerae (10) |
+ |
– |
– |
Vibrio parahaemolyticus (10) |
– |
+ |
– |
Vibrio vulnificus (10) |
– |
– |
+ |
Vibrio mimicus (2) |
– |
– |
– |
Vibrio alginolyticus (1) |
– |
– |
– |
ETEC ATCC 43896 |
– |
– |
– |
ETEC (5) |
– |
– |
– |
EHEC ATCC 43895 |
– |
– |
– |
EAEC (3) |
– |
– |
– |
Shigella spp. (2) |
– |
– |
– |
Shigella flexneri (3) |
– |
– |
– |
Shigella sonnei (2) |
– |
– |
– |
Salmonella spp. (2) |
– |
– |
– |
Salmonella enteritidis (2) |
– |
– |
– |
Salmonella typhimurium (2) |
– |
– |
– |
Table 2Target genes, primers, and probes used for multiplex real-time PCR detection of the three Vibrio spp
Vibrio spp. |
Primer or probe sequence (5′ to 3′) |
Target gene |
Amplicon size (bp) |
V. cholerae
|
VCF |
GCGTTGGGAGTGGCGTAA |
hlyA
|
57 |
VCR |
GGACTCGCCGCTGTAGACA |
|
|
VCP |
FAM-AGCACAGATGAATTGACCAMG-BNHQ |
|
|
V. parahaemolyticus
|
VPF |
AACCGTGGCGTTCCAGAA |
tlh
|
58p |
VPR |
CCGTCAAACGAATCAGTGCTT |
|
|
VPP |
VIC-TGAAAGCGGATTATGC-MGB |
|
|
V. vulnificus
|
VVF |
GATCGTTGTTTGACCGTAAACG |
vvhA
|
79p |
VVR |
TGCTAAGTTCGCACCACACTGT |
|
|
VVP |
NED-CAAAACGCTCACAGTCG-MGB |
|
|
Table 3Test results for the multiplex real-time PCR assay and isolation of Vibrio spp. from environmental samples
Vibrio spp. |
No. positive (%)
|
2007
|
2006
|
RT-PCR (n = 2,729) |
Isolates (n = 2,769) |
Isolates (n = 5,445) |
V. cholerae
|
330 (10.3) |
228 (5.6) |
213 (3.3) |
V. parahaemolyticus
|
2085 (64.7) |
1501 (37) |
1893 (29.3) |
V. vulnificus
|
621 (19.3) |
180 (4.4) |
106 (1.6) |