【作者】 龙飞；

【导师】 史贤明； 张忠明；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 博士

【摘要】 单核细胞增生李斯特菌(Listeria monocytogenes)是一种重要的食源性致病菌,广泛的分布于自然界,人体感染后的死亡率高达20%-30%。在美国每年约有2500起由单核细胞增生李斯特菌引起的食品中毒事件,其中致使患者死亡的高达500人(美国CDC),导致的经济损失高达23亿美元。近年来随着我国即食食品消费量的日益增加,单核细胞增生李斯特菌也逐渐成为威胁我国人民身体健康的主要食源性致病菌之一。因此,针对食品生产和消费中存在的单核细胞增生李斯特菌,建立一套快速、准确的检测方法非常必要。为解决传统PCR检测方法存在的不足,增加检测的准确性、可靠性和实用性,本文以单核细胞增生李斯特菌为研究对象,建立了一种广泛适用于食源性致病微生物检测的添加活菌内标的荧光定量PCR检测体系,可用于DNA提取前野生菌的准确定量和全程指示假阴性。主要研究内容和结果如下:1、四种细菌检测方法的比较:以单核细胞增生李斯特菌CMCC 54002作为待测菌株,比较了PCR检测方法与目前具有代表性的BIOLOG、VITEK和CRYSTAL3种微生物快速检测系统,并对本实验室保存的9株单核细胞增生李斯特菌做了血清学分型。结果表明:这3种微生物快速检测系统与PCR检测方法相比,其前处理过程时间长,需分离出纯的单菌落,并且都只能鉴定到李斯特菌属。PCR方法通过设计单核细胞增生李斯特菌种特异性引物,可快速的鉴定出单核细胞增生李斯特菌,并且无需纯化检测样品。通过对本实验室保存的9株单核细胞增生李斯特菌进行血清学分析,结果表明本实验室菌株涵盖了单核细胞增生李斯特菌主要的致病血清型(1/2a、4b、1/2c、3b)。2、扩增内标的设计:针对单核细胞增生李斯特菌主要毒力基因hly的保守序列,设计了一对特异性引物和相应的检测探针,并利用DNA序列随机改组软件,根据目标片断的序列特征,生成了1000组随机改组序列,然后通过荧光探针设计软件分析和BLAST-N比对筛选,找到了一条软件评分最高且与其它致病微生物基因组序列非同源的内标序列,该序列与目标片断具有相同长度(65 bp)和GC含量(55.4%),从而保证两者PCR扩增效率的一致性。3、活菌内标的构建:选用温敏型的穿梭质粒pKSV7来构建同源重组载体pKSV7-UIKD,该载体上连有上下游同源序列、扩增内标序列及卡那抗性基因,与质粒上自带的氯霉素抗性基因一起作为共同筛选标记,进行单核细胞增生李斯特菌双交换突变株的筛选,经过10轮培养和筛选,重组几率约为1.3%(4/300)。筛选出的双交换菌株用PCR、RT-PCR和测序分析进行了验证,结果表明成功获得了含扩增内标序列的hly基因缺失突变株LM-IAC,并作为活菌内标用于后续研究。4、准确测定野生菌DNA提取前菌体量的荧光定量PCR检测体系(LM-IACAQ-PCR assay)的建立:(1)评价了野生菌与活菌内标的DNA提取效率和PCR扩增效率,结果表明活菌内标与野生菌在10~9 cfu-10~5 cfu的范围内其DNA提取效率是相似的(约10%),同时两者具有相似的PCR扩增效率(野生菌1.05、活菌内标1.06)。(2)建立了根据活菌内标数量计算DNA提取前野生菌体量的线性回归方程y=-0.313x-0.0773,回归方程的相关系数R~2=0.9997,结果表明,根据该线性回归方程计算所得的DNA提取前野生菌菌体量与实际菌体含量基本一致,而由传统标准曲线法计算得到的菌体量要比实际量少一个数量级。5、全程指示假阴性的荧光定量PCR检测体系(LM-IAC EQ-PCR assay)的建立:(1)运用BIOLOG系统、VITEK系统比较了野生菌和活菌内标的生化特性,结果表明野生菌与活菌内标对检测板上的各项生化物质利用能力基本一致。(2)通过均匀设计试验评价了不同活菌内标加入量对野生菌在增菌培养中的影响,结果表明10~2 cfu-10~4 cfu的活菌内标接种量不影响野生菌的生长,其生长代时分别为49min(野生菌)和55 min(活菌内标)。(3)将10~3 cfu数量级的野生菌与活菌内标分别接入牛奶、鸡肉和腌肉样品,用UVM选择性增菌液培养,并以不添加食品样品的UVM纯培养为阳性对照,分别取增菌0 h、3 h、6 h的样品进行荧光PCR检测,结果表明阳性对照和牛奶样品中的野生菌和活菌内标不经过增菌培养即可检出,鸡肉样品经增菌3 h后野生菌和活菌内标方可检出,而腌肉样品要经增菌6 h后才可检出野生菌和活菌内标。这说明本研究建立的检测体系能够指示鸡肉和腌肉样品检测中的假阴性现象,有助于提高检测的准确率,同时可有效减少增菌时间。6、可用于单核细胞增生李斯特菌、沙门氏菌和副溶血弧菌普通PCR检测的多重扩增内标(multiple internal amplification control,mIAC)检测体系的建立:针对单核细胞增生李斯特菌hlyA基因、沙门氏菌invA基因和副溶血弧菌toxR基因设计特异性检测引物,根据这3对引物序列运用重叠PCR技术人工合成了1条多重扩增内标,可分别用于单核细胞增生李斯特菌、沙门氏菌和副溶血弧菌的普通PCR检测。含多重扩增内标的PCR检测体系的DNA检测灵敏度分别为:单核细胞增生李斯特菌,73.0 fg/μl;沙门氏菌,5.04 fg/μl和副溶血弧菌,76.4 fg/μl。通过对40份鸡肉、60份牛奶和60份虾样品分别进行单核细胞增生李斯特菌、沙门氏菌和副溶血弧菌的人工污染试验,结果显示鸡肉样品有2份出现假阴性、牛奶样品有2份出现假阴性、虾样品有3份出现假阴性,说明本研究建立的检测体系在进行大量样品检测时能够有效指示假阴性,有助于提高检测的准确率,并降低检测成本。更多还原

【Abstract】 Listeria monocytogenes is an important food-borne pathogen that causes listeriosis. It is widely distributed in the environment, and its infections can be life threatening, with a high fatality rate of 20 to 30%. It was estimated that approximately 2,500 individuals have developed listeriosis in the United States per year and the costs of the acute illness from foodborne Listeria are $2.3 billion. At the same time, L. monocytogenes has emerged as a significant foodborne pathogen in China during the past few years, along with a trend toward increasing consumption of ready-to-eat food products. Therefore, rapid detection methods are of significant importance to the L. monocytogenes quality control programs, which have to be applied throughout the food production chain.In order to overcome the limitations of traditional PCR method, a real-time quantitative PCR （Q-PCR） assay was developed with a mutant strain used as an internal control. This technique is suitable for most existing real-time PCR protocols, which can be used to quantify the initial amounts of L. monocytogenes in enriched samples and monitor the false negative results during the whole process. The main results are as follows:1. Four different bacterial detection methods （BIOLOG, VITEK, CRYSTAL and PCR） have been evaluated by L. monocytogenes CMCC 54002. The results showed that the PCR method is the best among these detection methods. The other three methods are time-consuming during sample preparation and request for separating the individual colonies from agar plates. Moreover, they can’t distinguish between L. monocytogenes and other Listeria species. In contrast, the PCR method can identify L. monocytogenes by specific primers and doesn’t need to purify the samples. Further serological analysis showed that the nine L. monocytogenes strains used in this study are serotypes 1/2a, 4b, 1/2c, 3b which are the main serotypes in most listeriosis cases.2. An IAC design method was developed by the computational DNA random shuffling. The hly gene was selected as a target gene for detecting L. monocytogenes by Q-PCR, and the conserved regions of the hly gene were used to design suitable L. monocytogenes-specific PCR primers and probe. Then, a DNA random shuffling software was used to shuffle the probe binding sequence. A total of 1000 random shuffling sequences were generated. Based on results from Bacon Designer 5.0 software analyses and BLAST-N searches, an IAC fragment that did not display significant similarity to any known pathogen DNA sequence was identified and synthesized by using the overlap-extension PCR technology. The IAC and hly amplicon share the equal length （65 bp） and GC content （55.4%）, which ensures the equal PCR amplification efficiencies.3. A mutant L. monocytogenes （LM-IAC） was obtained by exchanging the chromosomal hly gene with the IAC fragment through homologous recombination technology. The flanking regions of the hly gene, an IAC fragment and a kanamycin resistance gene were inserted into the pKSV7 shuttling vector, which contains a temperature sensitive replicon, to generate a recombinant vector called pKSV7-UIKD. A restoring strain was obtained by the replica plating method after continuous passages in BHI broth medium. Bacteria in colonies which could not grow on the chloramphenicol resistant plates, but can grow on the kanamycin resistant plates were candidates for the restoring strain. The recombination rate was about 1.3% （4/300）. The restoring strain was identified by PCR, RT-PCR and sequencing analysis to further confirmed that the IAC fragment and the kanamycin resistance gene had replaced the hly gene on the restoring strain chromosome.4. A LM-IAC AQ-PCR assay was developed for rapid and accurate detection of the original amount of wild-type L. monocytogenes before DNA isolation. （1） The amplification efficiencies and template extraction efficiency were estimated. The calculated amplification efficiencies of the hly amplicon and IAC were 1.05 and 1.06, respectively. The calculated template extraction efficiency results was similar （P > 0. 05） between wild-type L. monocytogenes and LM-IAC （about 10%） over a five orders of magnitude. （2） A calibration curve was established with the log （X/R） （X is the initial number of template, R is the initial number of bIAC） plotted againstΔC_T (C_TLM -C_TLM-IAC). The calibration curve calculated by linear regression was y = -0.313x - 0.0773 with a square regression coefficient of 0.9997. Comparison of the quantified wild-type L. monocytogenes contents with known amounts indicated that the established LM-IAC Q-PCR system was more reliable than the traditional absolute quantification method.5. A LM-IAC EQ-PCR assay was developed for monitoring the false negative results in the whole control. （1） The biochemical characteristics between wild-type L. monocytogenes and LM-bIAC have been compared with BIOLOG and VITEK’s identification system. The results showed that the two strains have similar biochemical characteristics. （2） The uniform design experiments were performed to analyze the comprehensive effects of different LM-IAC contents in medium on wild-type L. monocytogenes growth. It was found that the wild-type L. monocytogenes growth was stable with 10² cfu -10⁴ cfu LM-IAC contents. The generation time of wild-type L. monocytogenes and LM-IAC were 49 min and 55 min respectively. （3） Milk samples, chiken samples and pickle meat samples were artificially contaminated about 1000 cfu wild-type L. monocytogenes and LM-IAC and incubated with UVM selective enrichment broth, at the same time, preparing ddH₂O as positive control. Wild-type L. monocytogenes and LM-IAC which contaminated the ddH₂O and milk samples could be detected successfully by the LM-IAC Q-PCR assay without enrichment. However, wild-type L. monocytogenes and LM-IAC which contaminated the chicken and pickle meat samples could be detected after 3 h enrichment and 6 h enrichment respectively. It was demonstrated that the LM-IAC EQ-PCR assay could successfully eliminate the false-negative results between chicken and pickle meat samples. Therefore the advanteges of the LM-IAC EQ-PCR assay are accuracy and time- saving.6. A single multiple internal amplification control （mIAC） DNA molecule was constructed to detect the L. monocytogenes, Salmonella and Vibrio parahaemolyticus. Three pairs of primers （hlyAF/AR, invAF/ invar, toxRF/ toxRR） were designed based on the species-specific sequences of hlyA gene, invA gene and toxR gene respectively. The mIAC was synthesized by the overlap-extension PCR technology which contains the three pairs of primers’ sequences. Sensitivity of those mIAC PCR systems for purified target DNA were 73. 0 fg/μl （L. monocytogenes）, 5.04 fg/μl （Salmonella） and 76. 4 fg/μl （V. parahaemolyticus） respectively. In order to estimate the mIAC, 40 chiken samples, 60 milk samples and 60 shrimp samples were artificially contaminated with L. monocytogenes, Salmonella and Vibrio parahaemolyticus respectively. After PCR amplification, 3 chicken samples, 2 milk samples and 3 shrimp samples which turned out to be false-negative. The results demonstrated that the systems with mIAC could successfully eliminate the false-negative results.更多还原