【摘要】：徐懷恕等在1982年第一次提出了細(xì)菌“活的非可培養(yǎng)狀態(tài)(viable but nonculturable state,VBNC)”的概念,此后越來越多的報(bào)告闡明了細(xì)菌這種狀態(tài)的存在。所謂細(xì)菌的VBNC狀態(tài)是指長期處于不良環(huán)境條件下的細(xì)菌,用常規(guī)的固體平板培養(yǎng)方法不能培養(yǎng)出來,但仍然具有代謝活性的一種特殊生理狀態(tài)。處于該狀態(tài)的致病菌,在一定條件下可以復(fù)蘇,并具有潛在的致病性。VBNC狀態(tài)作為微生物學(xué)的一個(gè)全新概念,受到了極大的關(guān)注,它不僅對(duì)食品安全問題產(chǎn)生了新的考慮,而且對(duì)菌種保藏、活細(xì)菌的定義等都提出了新的探討。 副溶血弧菌是一種嗜鹽的革蘭氏陰性細(xì)菌,是大陸沿海地區(qū)食物中毒中最常見的一種病原菌。本文選用副溶血弧菌VP L4-90作為研究VBNC狀態(tài)的對(duì)象,探討了其進(jìn)入VBNC狀態(tài)的條件,并進(jìn)一步研究了VBNC狀態(tài)副溶血弧菌的復(fù)蘇現(xiàn)象及條件;建立了一種新型的基于PCR的活菌檢測(cè)方法,并將此方法運(yùn)用于高溫誘導(dǎo)的VBNC狀態(tài)副溶血弧菌的檢測(cè)中。 1.以常規(guī)的低溫貧營養(yǎng)方法誘導(dǎo)副溶血弧菌進(jìn)入VBNC狀態(tài),分別用AODC、DVC和PC等細(xì)菌檢測(cè)方法對(duì)誘導(dǎo)過程進(jìn)行檢測(cè),驗(yàn)證了副溶血弧菌能夠進(jìn)入VBNC狀態(tài);并對(duì)進(jìn)入VBNC狀態(tài)的副溶血弧菌的形態(tài)、遺傳物質(zhì)等進(jìn)行了初步研究。 2.進(jìn)一步對(duì)副溶血弧菌進(jìn)入VBNC狀態(tài)的條件(溫度、細(xì)菌的生長狀況和培養(yǎng)密度)進(jìn)行了優(yōu)化。結(jié)果發(fā)現(xiàn)穩(wěn)定期的副溶血弧菌VP L4-90在低溫貧營養(yǎng)條件下,較短的時(shí)間內(nèi)可以進(jìn)入VBNC狀態(tài);高溫下貧營養(yǎng)條件也能在短時(shí)間內(nèi)誘導(dǎo)副溶血弧菌進(jìn)入VBNC狀態(tài);培養(yǎng)密度只對(duì)副溶血弧菌VP L4-90進(jìn)入VBNC狀態(tài)的時(shí)間上有所影響。 3.在VBNC狀態(tài)細(xì)菌復(fù)蘇的研究方面,利用最大或然法定量樣品中可以復(fù)蘇的細(xì)菌數(shù)的結(jié)果說明,只有非常少數(shù)的VBNC狀態(tài)細(xì)菌具有復(fù)蘇的能力。復(fù)蘇后,少量的活菌可以利用死菌的營養(yǎng)進(jìn)行快速的生長。 4.本文利用EMA染料處理與Real Time PCR相結(jié)合,建立了一種新型的基于PCR的活菌檢測(cè)方法——EMA-PCR。 5.通過研究,發(fā)現(xiàn)在46℃副溶血弧菌一樣也可以進(jìn)入VBNC狀態(tài)。進(jìn)一步將EMA-PCR方法運(yùn)用于高溫誘導(dǎo)的實(shí)驗(yàn)中。雖然在高溫的培養(yǎng)過程中細(xì)菌出現(xiàn)了大量的死菌,但是其中也有小部分活的VBNC狀態(tài)存在,本實(shí)驗(yàn)結(jié)果給食品安全和細(xì)菌檢測(cè)提供了一個(gè)重要的參考。
[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，

本文編號(hào)：2311599