【作者】 祝儒刚；

【导师】 吕淑霞；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2011， 博士

【摘要】 副溶血弧菌(Vibrio parahaemolyticus, Vp)是一种革兰氏阴性嗜盐菌,广泛分布于海洋、河口环境,尤其是近海鱼类、贝类等海产品中。人类食用未煮熟或被致病性副溶血弧菌污染的海产品可引起急性胃肠炎,出现腹泻、呕吐、腹部痉挛等症状甚至死亡。近年来副溶血弧菌引发的食物中毒事件在日本、中国、新西兰、印尼、美国等不同国家和地区屡有发生,且其在沿海甚至内陆海产品中的分离率渐呈增长趋势。目前的研究认为,副溶血弧菌的毒力因子主要有粘附因子、侵袭力、溶血性毒素、尿素酶、脂多糖、胞外酶、三型分泌系统及摄铁系统等,其中溶血性毒素是副溶血弧菌最重要的毒力因子。溶血性毒素主要包括耐热直接溶血毒素(thermostabile direct hemolysin, TDH)、耐热直接相关溶血毒素(TDH-related hemolysin, TRH)和不耐热溶血毒素(thermolabile hemolysin, TLH),分别由相关的tdh、trh和tlh基因编码。TDH和TRH与副溶血弧菌的致病能力关系密切。临床分离的副溶血弧菌中超过90%为tdh阳性菌株,而环境中分离的副溶血弧菌只有1%-2%是tdh阳性株。本研究分别应用传统PCR技术和实时荧光定量PCR技术对海产品中致病性副溶血弧菌活细胞进行了定性和定量检测研究,建立了一种快速、灵敏并能够有效定量检测海产品中病原性副溶血弧菌活细胞的新方法。本研究分为三个部分：第一部分：首先分别用TZ裂解液法、煮沸法、CTAB/NaCl法和TENS法对模板DNA的提取进行优化,然后以细菌16SrDNA片段为扩增内标对照(internal amplification control, IAC),副溶血弧菌不耐热溶血毒素(thermolabile hemolysin,TLH)基因tlh和相对耐热直接溶血毒素(thermostable related hemolysin,TRH)基因trh为检测基因,设计引物,并优化多重PCR体系。特异性及灵敏度实验显示,该多重PCR体系检测致病性副溶血弧菌表现出极好的特异性。纯培养条件下,扩增内标存在时tlh基因和致病基因trh的检测灵敏度分别为1.3×102CFU/ml和1.3×103 CFU/ml;人工污染牡蛎样品,不经富集培养,扩增内标存在时tlh基因和致病基因trh的检测灵敏度分别为2.6×103 CFU/ml和2.6×104CFU/ml；经过6h的富集培养,tlh基因和trh基因的检测限均能达到2.6×102CFU/ml。结果表明,该检测体系特异性强、灵敏度高,并且扩增内标的存在可排除PCR检测致病性副溶血弧菌可能导致的假阴性结果,可提高检测的速度和精准度。第二部分：将一种DNA染料EMA (Ethidium bromide monoazide)与传统的PCR技术相结合,建立了一种能有效检测纯培养条件下副溶血弧菌死活菌细胞的新方法(EMA-PCR).研究结果表明,当用1.4μg/ml或更高浓度的EMA渗透处理含有4×108CFU/ml的副溶血弧菌死细胞菌悬液后再经20分钟曝光处理,其PCR结果呈阴性,而不经EMA处理的对照组其PCR结果则呈阳性；当EMA的用量等于或者小于6μg/ml时,副溶血弧菌活细胞的PCR扩增不会受到抑制。经EMA处理,含有不同比例的副溶血弧菌死细胞和活细胞的混合液中活的副溶血弧菌能够通过PCR被选择性的定量,最小的检测水平为10CFU/PCR。而且,研究发现在10～2×105CFU/PCR范围内,DNA相对荧光强度与死活细胞混合液中活细胞的对数具有线性关系。第三部分：对实时荧光定量PCR体系进行了优化,并将荧光染料EMA与RT-PCR (real-time PCR)技术相结合,建立了一种能选择性定量检测牡蛎中trh阳性副溶血弧菌活菌细胞的新方法。研究结果表明,使EMA成功插入死细胞DNA并且光解溶液中游离EMA的最佳曝光时间为20 min；不抑制副溶血弧菌活细胞DNA扩增的最大EMA浓度为2.0μg/ml;完全抑制热致死细胞DNA扩增的最小EMA浓度为1.4μg/ml。纯培养条件下,在22-2.2x107CFU范围内细胞数的对数值与Ct值之间呈严格的负相关性,并且不添加EMA时的检测灵敏度略大于添加EMA时的检测灵敏度,分别为22 CFU和2.2×102CFU。人工污染牡蛎样品,利用RT-PCR和EMA RT-PCR以及平板计数分别进行副溶血弧菌定量检测,结果表明EMA RT-PCR更接近于平板计数的结果,单纯RT-PCR定量的结果偏大。冻融实验表明,在温度小于55℃的水浴中对冷冻海产品进行解冻时,冻融过程对副溶血弧菌活细胞几乎没有影响。人工污染牡蛎样品,不经过富集,在4.7×102-4.7x106CFU范围内细胞数的对数值与Ct值之间呈严格的负相关性,并且人工污染牡蛎样品的RT-PCR检测限为4.7x102CFU,即人工污染牡蛎样品的RT-PCR检测灵敏度为94个活细胞/克牡蛎样品；经过6h的富集培养,纯培养和人工污染牡蛎样品中致病性副溶血弧菌活细胞的检测限均能达到47 CFU,纯培养与牡蛎样品的Ct值之间没有显著差异,Ct值与细胞数的对数之间也不存在线性关系。并且,在新采集样品的过夜富集培养液中背景微生物菌群总菌数为8.2×108CFU/ml时, EMA RT-PCR检测致病性副溶血弧菌灵敏度为4.7xl02CFU/ml,即就是说,经过夜富集培养的牡蛎样品培养液中,只要致病性副溶血弧菌活菌数大于等于4.7×102CFU/ml,就能够通过EMA RT-PCR方法检测出来。最后,在采集的45份海产品样品中,不经富集培养,利用EMA RT-PCR方法仅有一份牡蛎样品呈阳性,污染程度为114 CFU/g牡蛎样品。本研究为海产品中trh阳性副溶血弧菌活菌细胞的定性和定量检测提供了既快捷又准确的新方法,同时也为其它样品中活细胞的检测提供了新的思路。更多还原

【Abstract】 Vibrio parahaemolyticus is a Gram-negative halopilic bacterium distributed widely in the estuarine environment, especially in coastal fish, shellfish and seafood products. Pathogenic V. parahaemolyticus strains caused acute gastroenteritis, such as diarrhea, vomiting, abdominal cramps and so on, in humans after consumption of contaminated foods, most often involving unproperly cooked seafoods. In resent years, outbreaks of food poisoning caused by pathogenic V. parahaemolyticus have been reported in various geographic regions, including Japan, China, New Zealand, Indonesia and the USA. More recently, the occurrence of V. parahaemolyticus isolates from environments or seafoods not only in coastal areas but inland has shown an increasing tendency. The current research thinks that vibrio parahaemolyticus’s virulence factors are adhesion factor, aggressie, hemolytic toxins, urease, lipopolysaccharide, exocellular enzymes, secrete systemⅢand perturbation iron system etc, which hemolytic toxin is the most important virulence factors. Hemolytic toxin mainly includes thermostabile direct hemolysin, TDH-related hemolysin and thermolabile hemolysin, which are encoded by relevant gene of tdh, trh and tlh respectively. TDH and TRH have close relationship with the disease-causing ability of vibrio parahaemolyticus. More than 90% strains separated in clinic are TDH positive, however, TDH positive V. parahaemolyticus separated in environment is only 1% to 2%.Ttraditional PCR and real-time PCR technology to make the qualitative and quantitative study of of pathogen viable cells of Vibrio parahaemolyticus in seafood was conducted, and a fast, sensitive new method which could effectively identify and quantitative detection pathogen viable cells was developed. This research including three parts as follows:The first part:Firstly, template DNA extraction was optimized respectively using TZ lysing solution, boil, CTAB/NaCl and TENS, and 16SrDNA fragment of bacteria as internal amplification control(IAC) primers were designed to detect the genes of thermolabile hemolysin (tlh) and thermostable related hemolysin (trh) in vibrio parahaemolyticus, and the assay was optimized. The experiment of specificity and sensitivity showed that this multiplex PCR assay had a good specificity, and for pure culture, the detection limit of tlh and trh was 1.3×102 CFU/ml and 1.3×103 CFU/ml respectively in the presence of IAC; for samples of artificially contaminated oysters, however, the detection sensitivity of tlh and trh was 2.6×103 CFU/ml and 2.6×104 CFU/ml respectively in the presence of IAC without enrichment, and after 6 h enrichment as little as 2.6×102 CFU/ml of tlh and trh could be detected by this multiplex PCR. Results showed that the detection system had a good specificity and sensitivity. The existence of IAC could successfully eliminate false-negative results during using PCR technique to detect pathogenic vibrio parahaemolyticus and could improve the detection speed and accuracy.The second part:A new and efficient method for detection the viable and dead cell of pure cultured of vibrio parahaemolyticus was developed by using a DNA dye of ethidium bromide monoazide (EMA) in combination with the traditional polymerase chain reaction (EMA-PCR). The results showed that, under the light exposure for 20 minutes to photolyse the EMA in cell suspension of 4×108 CFU/ml vibrio parahaemolyticus treated with EMA at the concentration of 1.4μg/ml, the PCR results were negative, but the control without treatment by using EMA, the PCR results were positive. The PCR for viable cell of vibrio parahaemolyticus was not inhibited with the maximum concentration of EMA at 6μg/ml. After EMA treatment, the number of viable vibrio parahaemolyticus cells in varying ratios of viable to dead cells could be selectively quantified by PCR. The minimum level of detection was 10 CFU per PCR reaction. A linear relationship was found between the relative fluorescent intensity of the DNA bands and the log of genomic targets derived from the viable cells in mixtures of viable and dead cells in the range of 10 to 2×105 CFU per PCR reaction.The third part:Firstly the real-time fluorescence quantitative PCR system is optimized, and then a new method for selectively quantitative detection of trh-positive viable cells of Vibrio parahaemolyticus in oysters was developed using ethidium bromide monoazide (EMA) in combination with real-time PCR (RT-PCR, real-time polymerase chain reaction). The results showed that the optimized light exposure time to achieve crosslinking to DNA by the EMA in dead cells and to photolyse the free EMA in solution was 20 min. The EMA concentration of 2.0μg/ml or less did not inhibit the RT-PCR amplification of DNA derived from viable cells of Vibrio parahaemolyticus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 1.4μg/ml. For pure culture, there was a strict inverse correlation between the log of the number of cells and the associated Ct values in the range of 22～2.2×107 CFU, and the detection sensitivity of without EMA(22 CFU) was slightly higher than that of adding EMA(2.2×102 CFU). Artificial contamination of oyster samples, using RT-PCR, EMA RT-PCR and plate counts separately to quantitative detection of Vibrio parahaemolyticus, the analysis comparison indicated that the results of EMA RT-PCR was closer to plate counts and the number derived from RT-PCR was statistically higher than the number obtained from EMA RT-PCR and the plate count. The experiment of freezing and thawing showed that when freezed Oyster samples were thawed at below 55℃the freeze-thaw process had little effect on viable cells of V parahaemolyticus. Artificial contamination of oyster samples and without enrichment, there was a strict inverse correlation between the log of the number of cells and the associated Ct values in the range of 4.7×102～4.7×106 CFU, and the detection limit of the real-time PCR assay was 4.7×102 CFU for artificial contamination of Oyster samples, suggesting that the sensitivity of RT-PCR was 94 CFU/g oyster sample for artificial contamination. After 6 h enrichment, V. parahaemolyticus in pure cultures or Oyster homogenates containing an initial inoculum of 47 CFU, could be detected, and there was no significant difference in Ct values between pure cultures and Oyster homogenates. No linear relationship between the Ct values and log bacterial concentration was observed. The sensitivity of the real-time PCR detection of V. parahaemolyticus in the presence of background flora was examined by combining various dilutions of V. parahaemolyticus with an overnight enrichment culture generated using an uninoculated oyster sample. The total aerobic plate count from the overnight enrichment culture was 8.2×108 CFU/ml. Even in the presence of background flora, real-time PCR was capable of detecting as few as 4.7×102 CFU/ml of V parahaemolyticus. The real-time PCR method was able to detect the V. parahaemolyticus of more than 4.7×102 CFU/ml even in the presence of the background flora. Finally, a total of fourty-five oyster samples were tested for the trh positive strains of viable V. parahaemolyticus by real-time PCR after EMA treatment. Test results showed that only one oyster samples was trh positive of V. parahaemolyticus without enrichment, approximately at a mean bacteria concentration 114 CFU/g.This study provides both rapid and accurate new method for selectively qualitative and quantitative detection of trh-positive viable cells of Vibrio parahaemolyticus in seafoods, and also provides new ideals for detecting viable cells of other samples.更多还原