| BackgroundVibrio parahaemolyticus(Vp), which is one of the most important causative agents in food poisoning around the world, is recognized as the major cause of human gastroenteritis associated with consumption of seafood. This bacterium resides mainly in the marine environments and was frequently isolated from a variety of seafood including codfish, clam, octopus, shrimp, crab, lobster, scallop and oyster. Intake of raw or undercooked seafood, particularly shellfish, contaminated with Vibrio parahaemolyticus may lead to development of acute gastroenteritis characterized by diarrhea, headache, vomiting, nausea, abdominal cramps and low fever. It is a gram-negative, halophilic asporogenous rod that is straight or has a single, rigid curve. It has a single polar flagellum and could swim freely in liquid medium. The most suitable physiological environment for it was:pH 7.4～8.0, temperature 34～37℃, NaCl 2%～4%.The major virulence factors of Vibrio parahaemolyticus are thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). TDH is an enzyme that can lyse red blood cells on Wagatsuma blood agar plates(Kanagawa phemomenon,KP) and has functions as cardiotoxin, cytotoxicity, enterotoxin. TRH has similar immunogenicity, lethal effect and cytotoxicity though it couldn't cause Kanagawa phemomenon. There are nearly 67% similar amino acid sequences between TDH and TRH. Tdh and trh gene, which encode those two hemolysins respectively, locate in the chromosome of bacterium, have the length of 500～600bp and both of them can be detected by the molecular biological methods. Most Vibrio parahaemolyticus environmental strains were short of hemolysin genes, so this research discover the environmental strains which contain virulence genes.Since Vibrio parahaemolyticus was first recognized in Osaka in 1951, it has been reported to account for 20～30% of food poisoning cases in Japan. In Taiwan, Vibrio parahaemolyticus accounted for 69% of total bacterial food-borne outbreaks from 1981 to 2003. In China, it accounted for 31.1% of food-borne outbreaks between 1991 and 2001. Although the gastroenteritis caused by Vibrio parahaemolyticus is often self-limited, the infection may cause septicemia that is life-threatening to people who have liver disease or immune disorders. It is necessary to set up an accurate method for preventing the infection of Vp.At present, the major methods to detect Vp are as follows:1. Culture method. This method described in the National Standards Method(GB/T 4789.7-2008) is commonly used for the detection of Vibrio parahaemolyticus. First, samples were homogenated in alkaline saline peptone water for enrichment, and then spread plating on TCBS agar medium at 37℃for 24h. Typical blue-green colonies on TCBS were picked and identified by a series of biochemistry tests. Because this method has benefits of obtaining viable organism from the samples and certifying results through biochemical tests, it not only makes definite diagnosis in food poisoning, but also provide bacterial strains for scientific research. However, this method is labor-intensive and time-consuming. Moreover, Vp strains may be ignored because of careless colony picking, so it is not suitable for testing large numbers of samples.2. Serological method. This method was mainly used for classification of Vp after biochemical tests. Through the method, Vibrio parahaemolyticus were divided into 65 types on the basis of O antigen and K antigen, and it's important for the study on homology among different Vp strains around the world.3. Enzy-linked immunosorbent assay (ELISA). Either for double antibody sandwich method or for indirect ELISA, getting high quality of antibody is the key point to establish accurate diagnosis. These days people obtain polyclonal antibody mainly from animals immuned by inactivated strains or specific protein (TLH), and this polyclonal antibody is less effective in practical work. Preparation of high-quality monoclonal antibody will greatly increase the cost, that may partly explain why it's hard to see ELISA kits of Vp in market now.The development of molecular biology methods, which were considered to be significant all the time, was first reflected in updating technologies for Vp detection. In 1984, Kaper applied DNA hybridization to testing tdh gene in Vp strains. It is the first time to take advantage of DNA hybridization for Vp studies. In 1992, Nishibuchi first applied polymerase chain reaction (PCR) to detect virulence genes in Vp strains. This technology is classic, and still widely used in Vp detection up to now. In 2003, Real-time Fluorescent PCR (Real-time PCR) was used to detect tdh gene in Vp strains by Blackstone. In 2008, Yamazaki first used loop-mediated isothermal amplification (LAMP) technology to detect Vp. This new technology was recommended to be the screening tests for the Vp detection, for it's more convenient and needs fewer instruments than traditional technology. In addition, the target genes of Vp detection are renewal. For identification purposes, it is ideal to use a nucleotide sequece that is well conserved and that reflects the phylogenetic relationship. rRNA 3 sequence are often used for this purpose. However, rRNA sequence homologies between Vibrio parahaemolyticus and related species are so high that the rRNA does not appear to be suitable for the purpose described above. For example, the 16S rRNA sequences of Vibrio parahaemolyticus and Vibrio alginolyticus are >99% identical. Therefore, we should find a more reliable specific genes for Vp detection. According to many comparisons in different Vibrio sequences, the earliest specific gene we found was tlh gene, which encoding thermolabile hemolysin(TLH) in Vibrio parahemolyticus. After that, we continuously found pR72H, gyrB and toxR genes, and they were frequently used for Vp detection nowadays.Compared with other methods, molecular diagnosis has many advantages such as rapidity, simple operation and high sensitivity. Furthermore, many domestic enterprises have launched various Vp molecular diagnostic kits, which were easy to operate according to the manuals. We will evaluate the detective effect of them in this study.Objective1. To evaluate three kinds of kits for detection of Vibrio parahaemolyticus.2. To obtain genic information of Vp environmental strains in Guangzhou.Methods1. Cultivation and DNA extractionThe strains were inoculated to 3%NaCl alkaline peptone water (APW) at 37℃for 8h～18h, then bacterial DNA templates were extracted by the boiling method.2. Detection of the three kitsThree kinds of commercial kits were Vibrio parahaemolyticus PCR kit (CAT#91201A-50), Real-time PCR kit (Version Vp1) and LAMP kit (Version3, Sap.2009). Operation of each kit consults to the manuals.3. Sensitivities of the three kits To determine the sensitivity of each kit, serial 10-fold dilutions (from10-7 to 100) of Vibrio parahaemolyticus (Vp33847) culture broth were prepared. DNA templates were extracted from 1mL of each dilution by the boiling method, and the culture broth were quantified using the standard plating method..4. Specificities of the three kitsTo determine the specificity of each kit, strains including Vibrio parahaemolyticus (Vp33847, Vp14-90, Vp8-90, Vp25-9), Vibrio vulnificus ATCC27562, Staphylococcus flavus ATCC25923, Escherichia coli ATCC25922, Salmonella typhosa ST3216, Yersinia enterocoliticaWA, Laribacter hongkongensis LH679 were used in this study.5. Evaluation of the three kits using natural samplesSamples were collected from Huangsha Seafood Market in Guangzhou from July to September in 2010. All the samples were treated referring to the National Standard Method during sample preparation and strain storage.The three kits and the National Standard Method were used to detect Vp in samples, and then the detection rate of each method was calculated. The detection rates of four methods were compared using Cochran's Q test. Validity of each kit was evaluated by the results of the National standard method. Then, the whole processing time, the costs and the simplicity of operation in different kits were also compared.6. Genic information of environmental strainsGenomic DNA was extracted from Vp environmental strains, which were conformed by the National Standard Method. PCR technology was used to test the virulence genes, integronⅠandⅡ. The target bands by PCR were purified and then sent to be sequenced for verification.Results1. Sensitivities of the three kits The limitis of detection for Vibrio parahamolyticus PCR kit, real-time PCR kit and LAMP kit were 1.18 103 cfu/mL,11.8 cfu/mL and 11.8 cfu/mL respectively.2. Specificities of the three kitsAccording to detection results by PCR kit, real-time PCR kit and LAMP kit, all Vibrio parahaemolyticus strains showed positive results, while other strains showed negetive results.3. Evaluation of the three kits using natrual samplesIn this study, we collected 205 seafood-samples including 25 Penacus orientalises,70 Scallops,58 clams and 52 Sinonovacula constrictas. The detection rate of the National Standard Method was 31.7% and 65 positive samples were found in all the samples. The detection rate of PCR kit was 63.9% and 131 positive samples were found. The detection rate of Real-time PCR kit was 66.8% and 137 positive samples were found. The detection rate of LAMP kit was 65.9% and 135 positive samples were found.The detection rates of the three kits and the National Standard Method were compared by Cochran's Q test (χ2=189.078, P<0.001). There was signficant difference among them. Compared with the National Standard Method, higher detection rates were observed from the three kits. When the detection rates of three kits were compared with each other, no significant difference was observed(χ2=5.091, P=0.78).Compared with the National Standard Method, the sensitivities of PCR kit, real-time PCR kit and LAMP kit were all 100%; the specificities were 52.9%,48.6% and 50.0% respectively; the false negative rates were all 0%; the false positive rates were 47.1%,51.4% and 50.0% respectively; the Youden's indices were 0.53,0.49 and 0.50 respectively; the agreement rates were 67.8%,64.9% and 65.9% respectively; the Kappa values were 0.416,0.375 and 0.388 respectively; the positive predictive values were 49.6%,47.4% and 48.1% respectively; the negative predictive values were all 100%.The processing time of PCR kit, real-time PCR kit and LAMP kit were 120min, 80min and 80min in turn, and the costs were￥10,￥40 and￥45 respectively for a single sample. The LAMP kit was the simplest to operate, and the PCR kit was the most difficult one.4. Genic information of isolated Vp strainsNo trh gene, integronⅠandⅡwere observed in all 65 environmental strains in this research. Only 1 strain contains tdh gene and the sequencing result showed it belonged to tdh2 gene.Conclusion1. The detection rates of the three kits were all higher than that of the National Standard Method, and yet no significant difference was observed among detection rates of the three kits.2. The three kinds of commercial kits were all characterized by high sensitivity, simple operation and less time-consuming. Especially, the LAMP kit was suitable for primary units because it needs fewer instruments. Therefore, they were recommended to be the screening tests for the detection of Vibrio parahaemolyticus.3. Tdh positive strains from seafood may be the potential pathogen for food poisoning.4. This study suggested integronⅠand integronⅡseldom existed in environmental Vp strains. |
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