發(fā)布時(shí)間：2018-07-17 00:46

【摘要】：阪崎腸桿菌(Enterbacter sakazakii),是寄生在人和動(dòng)物腸道內(nèi)的一種革蘭氏陰性無芽孢桿菌,周身鞭毛、能運(yùn)動(dòng)、兼性厭氧。該菌能引起嚴(yán)重的腦膜炎、壞死性小腸結(jié)腸炎和菌血癥等病癥,可感染各年齡段免疫力低下的人群,尤其是新生兒、早產(chǎn)兒及出生時(shí)身體條件較差的嬰兒,感染后嬰兒死亡率高達(dá)50%以上,是一種重要的食源性條件致病菌。近年來,國內(nèi)外越來越多的研究表明:嬰幼兒配方奶粉是引起嬰幼兒感染的主要傳染源和傳播媒介。目前,GB法檢測阪崎腸桿菌主要是基于傳統(tǒng)的微生物培養(yǎng),然后綜合菌落形態(tài)、生化特征等進(jìn)行鑒定。此法操作繁瑣、耗時(shí)耗力、且不易分辨結(jié)果,存在一定假陽性。所以近年來,采用分子生物學(xué)的方法檢測食源性病菌得到越來越廣泛的應(yīng)用。本論文基于中華人民共和國出入境檢驗(yàn)檢疫行業(yè)標(biāo)準(zhǔn)(SN/T 1632.3-2013)中阪崎腸桿菌(克羅諾桿菌屬)實(shí)時(shí)熒光定量PCR檢驗(yàn)方法,首先將PCR反應(yīng)各組分預(yù)混成Mix Buffer,大大簡化了操作步驟,同時(shí)對(duì)各組分濃度及反應(yīng)體系進(jìn)行了優(yōu)化,實(shí)現(xiàn)阪崎腸桿菌Real-time PCR的快速檢測。其次,通過對(duì)比酚氯仿和加熱裂解兩種DNA提取方法,對(duì)加熱裂解法進(jìn)行了優(yōu)化,并用貝因美奶粉樣本和人工污染樣本進(jìn)行了驗(yàn)證。人工污染奶粉的阪崎腸桿菌的檢出限可達(dá)4.7× 103CFU/ml。本文中所有實(shí)時(shí)熒光定量PCR實(shí)驗(yàn)都是在博日公司自主研發(fā)的實(shí)時(shí)熒光定量PCR儀器平臺(tái)和博日專利產(chǎn)品“標(biāo)準(zhǔn)PCR實(shí)驗(yàn)室”內(nèi)完成,從而初步建立了“試劑、儀器、軟件、PCR污染防控”為一體的阪崎腸桿菌快速檢測系統(tǒng),為客戶提供從硬件到軟件,從實(shí)驗(yàn)操作到環(huán)境污染控制的整體解決方案,實(shí)現(xiàn)“穩(wěn)定”的快速檢測。
[Abstract]:Enterbacter sakazakii), is a gram-negative bacillus that is parasitic in human and animal intestines. The bacteria can cause severe meningitis, necrotizing enterocolitis and bacteremia, and can infect people of all ages with low immunity, especially newborns, premature infants and babies with poor physical conditions at birth. The infant mortality rate after infection is over 50%, which is an important food-borne conditional pathogen. In recent years, more and more studies at home and abroad show that infant formula is the main source of infection and transmission media. At present, the detection of Enterobacter sakazakii by GB method is mainly based on the traditional microbial culture, and then comprehensive colony morphology, biochemical characteristics and so on. This method is complicated, time-consuming, and difficult to distinguish the results, there is a certain false positive. Therefore, molecular biology has been used to detect foodborne bacteria more and more widely in recent years. Based on the real-time fluorescence quantitative PCR method of Enterobacter sakazakii (Crotonia) in the Entry-Exit Inspection and Quarantine Industry Standard of the people's Republic of China (SNR / T 1632.3-2013), the PCR reaction components are premixed into mix buffer, which greatly simplifies the operation procedure. At the same time, the concentration of each component and reaction system were optimized to realize the rapid detection of Enterobacter sakazakii Real-time PCR. Secondly, by comparing phenol chloroform and heating cracking two DNA extraction methods, the heating pyrolysis method was optimized and verified by Beimei milk powder samples and artificially contaminated samples. The detection limit of Enterobacter sakazakii in artificially contaminated milk powder was 4.7 脳 103 CFU / ml. In this paper, all the real-time fluorescent quantitative PCR experiments were carried out in the real-time fluorescent quantitative PCR instrument platform developed by Boji Company and the standard PCR laboratory, which is the patented product of Boji Company. Thus, the "reagent, instrument" was preliminarily established. The rapid detection system of Enterobacter sakazakii which is integrated with PCR pollution prevention and control software provides the whole solution from hardware to software, from experimental operation to environmental pollution control, and realizes the "stable" rapid detection of Enterobacter sakazakii.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS252.51;O657.3

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