Development of quantitative real-time polymerase chain reaction for the detection of Vibrio vulnificus based on hemolysin (vvhA) coding system.
OBJECTIVE
To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene (vvhA) coding cytolysin.


METHODS
Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles (Ct value) and fluorescence intensity enhancement (DeltaRn value) were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples.


RESULTS
The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 10(3) copies with a Ct value of 37.94+/-0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus.


CONCLUSIONS
TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.
Department of Medical Microbiology and Parasitology, College ofMedicine, Zhejiang University, Hangzhou 310058, Zhejiang, China. wuzenhuil@yahoo.com.cn
Zeng-Hui
ZH
Wu
Yong-Liang
YL
Lou
Yi-Yu
YY
Lu
Jie
J
Yan
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D051379:Mice; D008813:Mice, Inbred ICR; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D041261:Vibrio vulnificus / Q000302:isolation & purification*
D001426:Bacterial Proteins; D017931:DNA Primers; C470630:VvhA protein, Vibrio vulnificus