副溶血弧菌VPL4-90活的非可培养状态的初步研究
发布时间：2018-11-05 09:09
【摘要】：徐怀恕等在1982年第一次提出了细菌“活的非可培养状态(viable but nonculturable state,VBNC)”的概念,此后越来越多的报告阐明了细菌这种状态的存在。所谓细菌的VBNC状态是指长期处于不良环境条件下的细菌,用常规的固体平板培养方法不能培养出来,但仍然具有代谢活性的一种特殊生理状态。处于该状态的致病菌,在一定条件下可以复苏,并具有潜在的致病性。VBNC状态作为微生物学的一个全新概念,受到了极大的关注,它不仅对食品安全问题产生了新的考虑,而且对菌种保藏、活细菌的定义等都提出了新的探讨。 副溶血弧菌是一种嗜盐的革兰氏阴性细菌,是大陆沿海地区食物中毒中最常见的一种病原菌。本文选用副溶血弧菌VP L4-90作为研究VBNC状态的对象,探讨了其进入VBNC状态的条件,并进一步研究了VBNC状态副溶血弧菌的复苏现象及条件;建立了一种新型的基于PCR的活菌检测方法,并将此方法运用于高温诱导的VBNC状态副溶血弧菌的检测中。 1.以常规的低温贫营养方法诱导副溶血弧菌进入VBNC状态,分别用AODC、DVC和PC等细菌检测方法对诱导过程进行检测,验证了副溶血弧菌能够进入VBNC状态;并对进入VBNC状态的副溶血弧菌的形态、遗传物质等进行了初步研究。 2.进一步对副溶血弧菌进入VBNC状态的条件(温度、细菌的生长状况和培养密度)进行了优化。结果发现稳定期的副溶血弧菌VP L4-90在低温贫营养条件下,较短的时间内可以进入VBNC状态;高温下贫营养条件也能在短时间内诱导副溶血弧菌进入VBNC状态;培养密度只对副溶血弧菌VP L4-90进入VBNC状态的时间上有所影响。 3.在VBNC状态细菌复苏的研究方面,利用最大或然法定量样品中可以复苏的细菌数的结果说明,只有非常少数的VBNC状态细菌具有复苏的能力。复苏后,少量的活菌可以利用死菌的营养进行快速的生长。 4.本文利用EMA染料处理与Real Time PCR相结合,建立了一种新型的基于PCR的活菌检测方法——EMA-PCR。 5.通过研究,发现在46℃副溶血弧菌一样也可以进入VBNC状态。进一步将EMA-PCR方法运用于高温诱导的实验中。虽然在高温的培养过程中细菌出现了大量的死菌,但是其中也有小部分活的VBNC状态存在,本实验结果给食品安全和细菌检测提供了一个重要的参考。
[Abstract]:In 1982, Xu Huai Shu et al first proposed the concept of "living non-culturable state (viable but nonculturable state,VBNC". Since then, more and more reports have clarified the existence of bacteria in this state. The VBNC state of bacteria is a kind of special physiological state which can not be cultured by conventional solid plate culture method but still has metabolic activity. The pathogenic bacteria in this state can be resuscitated under certain conditions and have potential pathogenicity. As a new concept of microbiology, VBNC state has received great attention. Moreover, the preservation of bacteria and the definition of live bacteria are also discussed. Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a halophilic gram-negative bacterium and the most common pathogen of food poisoning in coastal areas of China. In this paper, Vibrio parahaemolyticus VP L4-90 was selected as the object to study the state of VBNC, and the conditions for its entry into VBNC state were discussed, and the resuscitation phenomena and conditions of Vibrio parahaemolyticus in VBNC state were further studied. A new method based on PCR for the detection of Vibrio parahaemolyticus was established and applied to the detection of Vibrio parahaemolyticus in VBNC state induced by high temperature. 1. Vibrio parahaemolyticus (Vibrio parahaemolyticus) was induced into VBNC state by conventional low temperature poor nutrition method. The induced process was detected by AODC,DVC and PC respectively. The results showed that Vibrio parahaemolyticus could enter VBNC state. The morphology and genetic material of Vibrio parahaemolyticus (Vibrio parahaemolyticus) in VBNC state were studied. 2. The conditions for the entry of Vibrio parahaemolyticus into VBNC state (temperature, bacterial growth and culture density) were further optimized. The results showed that the stable vibrio parahaemolyticus VP L4-90 could enter the VBNC state in a short time under the condition of low temperature and poor nutrition, and could also induce Vibrio parahaemolyticus to enter the VBNC state in a short time under the condition of high temperature. The culture density only affected the time of VP L 4-90 entry into VBNC state of Vibrio parahaemolyticus. 3. In the research of VBNC state bacteria resuscitation, using the results of the most probable amount of samples which can be resuscitated, it shows that only a very few VBNC state bacteria have the ability of resuscitation. After resuscitation, a small number of live bacteria can use the nutrition of dead bacteria for rapid growth. 4. In this paper, a new detection method of live bacteria based on PCR, EMA-PCR., was established by combining EMA dye treatment with Real Time PCR. 5. It was found that Vibrio parahaemolyticus could enter VBNC state at 46 鈩，
本文编号：2311599
[Abstract]:In 1982, Xu Huai Shu et al first proposed the concept of "living non-culturable state (viable but nonculturable state,VBNC". Since then, more and more reports have clarified the existence of bacteria in this state. The VBNC state of bacteria is a kind of special physiological state which can not be cultured by conventional solid plate culture method but still has metabolic activity. The pathogenic bacteria in this state can be resuscitated under certain conditions and have potential pathogenicity. As a new concept of microbiology, VBNC state has received great attention. Moreover, the preservation of bacteria and the definition of live bacteria are also discussed. Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a halophilic gram-negative bacterium and the most common pathogen of food poisoning in coastal areas of China. In this paper, Vibrio parahaemolyticus VP L4-90 was selected as the object to study the state of VBNC, and the conditions for its entry into VBNC state were discussed, and the resuscitation phenomena and conditions of Vibrio parahaemolyticus in VBNC state were further studied. A new method based on PCR for the detection of Vibrio parahaemolyticus was established and applied to the detection of Vibrio parahaemolyticus in VBNC state induced by high temperature. 1. Vibrio parahaemolyticus (Vibrio parahaemolyticus) was induced into VBNC state by conventional low temperature poor nutrition method. The induced process was detected by AODC,DVC and PC respectively. The results showed that Vibrio parahaemolyticus could enter VBNC state. The morphology and genetic material of Vibrio parahaemolyticus (Vibrio parahaemolyticus) in VBNC state were studied. 2. The conditions for the entry of Vibrio parahaemolyticus into VBNC state (temperature, bacterial growth and culture density) were further optimized. The results showed that the stable vibrio parahaemolyticus VP L4-90 could enter the VBNC state in a short time under the condition of low temperature and poor nutrition, and could also induce Vibrio parahaemolyticus to enter the VBNC state in a short time under the condition of high temperature. The culture density only affected the time of VP L 4-90 entry into VBNC state of Vibrio parahaemolyticus. 3. In the research of VBNC state bacteria resuscitation, using the results of the most probable amount of samples which can be resuscitated, it shows that only a very few VBNC state bacteria have the ability of resuscitation. After resuscitation, a small number of live bacteria can use the nutrition of dead bacteria for rapid growth. 4. In this paper, a new detection method of live bacteria based on PCR, EMA-PCR., was established by combining EMA dye treatment with Real Time PCR. 5. It was found that Vibrio parahaemolyticus could enter VBNC state at 46 鈩，
本文编号：2311599
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