| Background and Objective Vibrio vulnificus is a Gram-negtice bacterium thatis found in estuarine environment around the world in areas with warm seawater temperature (>20℃) and frequently contaminates seafood. This organism can produce serious disease in humans and eels. Ingestion of raw contaminated seafood with V. vulnificus can result in septicemia. Contamination of wounds with the microbe will cause devastating necrotizing fasciitis, which can progress to septicemia. Additionally, V. vulnificus has been reported to cause gastroenreritis and airway obstruction. In predisposed people with underlying conditions such as hemochromatosis, alcoholism and liver cirrhosis, the mortality rate for wound infections derived from this bacterium are often as high as 50% and for septicemia can be as high as 75%. V. vulnificus is the leading cause of reported death from seafood in the United States with approximately 40 cases reported each year. Numerous outbreaks of V. vulnificus infections derived from seafood consumption also have been reported in Asia and Europe. In China, most reported cases occurredin coastal areas. For example, the estimated prevalence of V. vulnificus infection in Taiwan was 0.606～1.237/10~6 persons from 1996 to 2000 and the mortality rate of the disease was about 41%. Wound infections may start as redness and swelling at the site of the wound that can progress to affect the whole body rapidly, sometimes the aggressive surgical treatment of amputation of the affected limb is necessary to prevent death. Due to the acute development of disease, there is a need for the rapid detection of V. vulnificus in clinical laboratory.V. vulnificus belongs to the fifth cluster of Vibrio which can be classed three biotypes, type I and III produces disease in human; type II can cause disease in eel. The cytolysin haemolysin (vvhA) gene is present in almost the whole wild-type of V. vulnificus strains and is known as a species-specific gene of V. vulnificus. So this gene is an ideal target to design the primers and probe for the conventional PCR and real-time PCR.The TaqMan assay is widely used, as it is a highly sensitive, specific and quantitative PCR detection system. This method allows detection of PCR product during amplification and decreases the two main problems of conventional PCR: risk of gel electrophoresis of PCR products and post-PCR contamination. Thus it is suitable for the detection of many infectious pathogens in clinical specimens. On the basis of detection by the method of conventional PCR, the TaqMan real-time PCR assay was established to guide the clinical to reduce the amputation and mortality of patients infected with V.vulnificus.Material and Methods The bacterial strains of the various non-Vibrio vulnificusspecies used in this study were from Zhejiang University School of Medicine and V. vulnificus was isolated from the clinical sample of Wenzhou Medical College. Bacterial cultures were centrifuge and pellets were colleted for DNA extraction with a DNA minikit (Takara) according to the manufacturer's instruction. The primersand probe were located at the conserved regions of cytolysin haemolysin (vvhA) gene, designed by the software of Primer Express. The probe was labeled at the 5' end with the reporter dye (FAM) and at the 3'end with the quencher dye (TAMRA). The targent DNA amplification were purified with 3S PCR Product Purification kit V2.0 (SNBC) and ligated to the pMD19-T vectors. The recombinant pMD19-T vector named as pMD19-T-vvhA100 and transferred into E.coli DH5α. The obtained positive colonies by blue-white screening were amplified and then the plasmid in the bacteria were respectively extracted by alkaline-denature method. In order to detect the sensitivity of the TaqMan assay, the purified plasmid DNA was serially diluted to final concentrations of 10~7～10~2 copies per PCR tube and analyzed with the real-time PCR. In order to determine the specificity of primers and probe, a number of non-Vibrio (Vibrio cholerae, Escherichia coli, Eberthella typhi, Bacillus aeruginosus, Bacterium shigae, Enterobacter aerogenes, Staphylococcus aureus and Bacillus proteus) and Vibrio strains were tested by vvhA TaqMan real-time PCR. Due to the difficulty to obtain the clinical samples of patients infected with V.vulnificus, the mice model infected with V.vulnificus was established to imitate the diseases caused by V.vulnificus. 1×10~5CFU, 1×10~6CFU and 1 × 10~7CFU of V. vulnificus was used to infect mice. In each experiment there were four groups, a control group treated with nutrient solution, and three other groups treated with injection into the right thigh of mouse, enterocoelia, and oral inoculation respectively that each having 6 mice. In order to evaluate the TaqMan assay developed in this study for V. vulnificus, The DNA of samples of mice tissue contaminated V. vulnificus was extracted as the template for the TaqMan real-time assay.Results the cytolysin haemolysin (vvhA) gene can be well amplified by primers. The DNA sequence of amplified fragment was determined by Invitrogen Company.Compared with the reported nucleotide sequences in the Gene Bank, the similarity for the nucleotide sequence of a 100-bp fragment in the vvhA gene was 100% identical. In order to acquire the positive control of TaqMan assay to detect the V. vulnificus, the recombinant pMD19-T-vvhA100 vector containing the amplified fragment of vvhA gene was established. The positive control of recombinant pMD19-T-vvhA100 vector acquired good results in experiment. In order to determine the specificity of primers and probe, DNA extraction from a number of Vibrio vulnificus and non-Vibrio vulnificus (Vibrio cholerae, Escherichia coli, Eberthella typhi, Bacillus aeruginosus, Bacterium shigae, Enterobacter aerogenes, Staphylococcus aureus and Bacillus proteus) were tested using the TaqMan real-time PCR assay. As expected no cross-reactivity was observed with any of non-Vibrio tested. For the aim of enhancing the sensitivity and efficiency of amplification namely acquiring the minimum of Ct value correspondingly the maximum of ΔRn value, the reaction conditions were optimized by varying primer and probe concentrations to obtain optimal fluorescent signal accumulation and rapid measurement of Ct values. Optimization was carried out by methodical variation of each test parameter under standard PCR conditions. The result show that after optimized, the Ct value decreased 5.51 and the ARn value rose from 0.6 to 1.1. The sensitivity of the TaqMan real-time PCR assay was as high as 0.01 ng of V. vulnificus DNA or 10~3 copies of pMD19-vvhA100. The SD values of the detection results repeated for three times using pMD19-vvhA100 with different concentrations were lease than 0.79, the conventional PCR only can detect the 1ng of V. vulnificus DNA or 10~5 copies of pMD 19-vvhA100.The mice of groups treated with injecting into the area over the right thigh of mouse, injecting into the enterocoelia, were died in 12 hours (mice groups treated with injection into the right thigh of mouse and the enterocoelia died in 12 hours)and the groups treated with oral inculation were alive in 12 hours. The samples of control group and oral inculation groups were negative by the TaqMan real-time PCR. The samples of blood and subcutaneous tissue from the groups treated with injecting into the area over the right thigh of mouse, and groups treated with injecting into the enterocoelia were positive by the method of TaqMan real-time PCR.Conclusion The TaqMan real-time fluorescent quantitative PCR established in this study for V.vulnificus vvhA gene detection has advantages such as quickness, stability, sensitivity and specificity, indicating this method can be used for clinical laboratory diagnosis of septicemia and wound infection caused by V. vulnificus.
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