本文選題：單核細(xì)胞增生性李斯特菌　切入點(diǎn)：環(huán)介導(dǎo)等溫?cái)U(kuò)增　出處：《吉林大學(xué)》2017年碩士論文

【摘要】：單核細(xì)胞增生性李斯特菌是一種食源性人獸共患病的革蘭氏陽(yáng)性病原菌,可引起人和多種動(dòng)物罹患李斯特菌病。早在20世紀(jì)90年代,它已被列為四種食源性致病菌之一,近年來(lái),單核細(xì)胞增生性李斯特菌感染病例在世界范圍內(nèi)呈上升趨勢(shì)。目前,該細(xì)菌的傳統(tǒng)檢測(cè)方法,操作流程復(fù)雜,檢測(cè)周期長(zhǎng),難以滿足快速檢測(cè)食品所需效率。因此,有必要建立一種快速、方便的單核細(xì)胞增生性李斯特菌的檢測(cè)方法。本研究針對(duì)單核細(xì)胞增生性李斯特菌(以下簡(jiǎn)稱單增李斯特菌)hly A基因設(shè)計(jì)了4條特異性的環(huán)介導(dǎo)等溫核酸擴(kuò)增(loop-mediated isothermal amplification,LAMP)引物,通過(guò)篩選引物、優(yōu)化反應(yīng)條件,建立了單增李斯特菌的LAMP快速檢測(cè)方法。試驗(yàn)結(jié)果顯示,該方法能夠特異地檢測(cè)單增李斯特菌,其最低檢測(cè)限為3 CFU/m L。擴(kuò)增產(chǎn)物可通過(guò)瓊脂糖凝膠電泳、顯色反應(yīng)進(jìn)行判定。LAMP可以快速、直觀、準(zhǔn)確地檢測(cè)單增李斯特菌,是一種適合基層現(xiàn)場(chǎng)應(yīng)用的檢測(cè)方法。在此基礎(chǔ)上,研制組裝的單增李斯特菌快速檢測(cè)試劑盒,63℃,45min內(nèi)即可檢出單增李斯特菌DNA的擴(kuò)增產(chǎn)物,其最低檢測(cè)限為3 CFU/m L,特異性為100%,無(wú)假陽(yáng)性和假陰性出現(xiàn)。采用20份樣品,進(jìn)行批內(nèi)批間重復(fù)性試驗(yàn)、效期穩(wěn)定性試驗(yàn)等驗(yàn)證,結(jié)果表明本試劑盒準(zhǔn)確、穩(wěn)定、重現(xiàn)性好,且LAMP檢測(cè)中包括前期樣品處理、DNA提純等全部過(guò)程僅在2h內(nèi)便能完成。本課題還在所研究的環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)基礎(chǔ)上,結(jié)合橫向流動(dòng)試紙條技術(shù)(Lateral flow dipstick,LFD)原理,設(shè)計(jì)出一種便捷、快速的檢測(cè)單增李斯特菌的方法。研究過(guò)程中,針對(duì)單增李斯特菌hly A基因設(shè)計(jì)4條特異性引物和1條異硫氰酸熒光素(Fluorescein isothiocyanate,FITC)標(biāo)記的探針。根據(jù)這種方法檢測(cè)發(fā)現(xiàn),生物素標(biāo)記LAMP擴(kuò)增產(chǎn)物可以特異性地與FITC標(biāo)記的探針雜交,雜交產(chǎn)物經(jīng)LFD檢測(cè)。LAMP體系經(jīng)優(yōu)化后,其擴(kuò)增溫度為63℃,反應(yīng)時(shí)間50min,從樣品處理直到檢測(cè)完成僅需80min。LAMP-LFD方法可特異性地檢出單增李斯特菌,對(duì)其它6株常見食源性致病菌的檢測(cè)結(jié)果均顯示陰性;對(duì)純細(xì)菌培養(yǎng)物的檢測(cè)靈敏度為3.6 CFU/m L,是利用外引物建立的PCR方法的100倍�？傊�,用LAMP-LFD方法檢測(cè)單增李斯特菌,節(jié)約環(huán)保,流程簡(jiǎn)單,結(jié)果客觀明了,適合醫(yī)院、養(yǎng)殖中心等場(chǎng)所等進(jìn)行實(shí)時(shí)監(jiān)測(cè),值得廣泛推廣。
[Abstract]:Listeria monocytogenes (Listeria monocytogenes) is a Gram-positive pathogen of zoonosis of human and animal, which can cause Listeria disease in human and many animals.As early as 1990s, it has been listed as one of the four foodborne pathogenic bacteria. In recent years, the infection of Listeria monocytogenes is on the rise in the world.At present, the traditional method of bacterial detection is complex in operation and long in detection period, so it is difficult to meet the need efficiency of rapid detection of food.Therefore, it is necessary to establish a rapid and convenient method for detection of Listeria monocytogenes.In this study, four specific loop-mediated isothermal amplification amplification primers were designed for Listeria monocytogenes (hereinafter referred to as Listeria monocytogenes) gene, and the reaction conditions were optimized by screening primers.A rapid LAMP detection method for Listeria monocytogenes was established.The results showed that the method could specifically detect Listeria monocytogenes with a detection limit of 3 CFU/m L.Amplification products can be determined by agarose gel electrophoresis and color reaction. Lamp can be used to detect Listeria monocytogenes quickly, intuitively and accurately. It is a suitable method for detection of Listeria monocytogenes in the field.On the basis of this, the amplified products of Listeria monocytogenes DNA could be detected within 45 minutes at 63 鈩，

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