莖環(huán)型引物—環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)檢測(cè)奶粉中單增李斯特菌的研究
發(fā)布時(shí)間：2018-07-14 11:09
【摘要】：單核細(xì)胞增生李斯特氏菌(Listeria monocytogenes,LM)簡(jiǎn)稱(chēng)單增李斯特菌,是一種能夠引起人畜共患病的食源性致病菌,屬李斯特菌屬。廣泛分布于自然界,食用了被單增李斯特污染的食物,可導(dǎo)致人和動(dòng)物敗血癥、流產(chǎn)、腦膜炎等,病死率高達(dá)30%~70%,是人類(lèi)最重要的食源性病原菌之一。單增李斯特菌的檢測(cè)、鑒定以及防控技術(shù)已經(jīng)受到極大的關(guān)注,其檢測(cè)方法一直沿用過(guò)去傳統(tǒng)的分離鑒定方法,己不能適應(yīng)現(xiàn)代快速檢測(cè)的要求。 環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)(Loop-mediated isothermal amplification, LAMP)是一種新穎的核酸體外擴(kuò)增技術(shù),具有簡(jiǎn)單、快速、敏感、特異性強(qiáng)的特點(diǎn),已經(jīng)應(yīng)用于檢測(cè)病原菌等諸多領(lǐng)域。該方法自身最大的局限性就是易出現(xiàn)假陽(yáng)性。本研究利用LAMP技術(shù)檢測(cè)單增李斯特菌,對(duì)LAMP引物進(jìn)行設(shè)計(jì)、篩選及改進(jìn),選擇帶有一定莖環(huán)結(jié)構(gòu)的引物,避免由于多條引物在同一條件下擴(kuò)增,可能互補(bǔ)擴(kuò)增而出現(xiàn)的假陽(yáng)性,進(jìn)一步提高LAMP檢測(cè)的準(zhǔn)確性,可稱(chēng)之為莖環(huán)型引物-LAMP(Stemloop-Primer Loop-mediated Isothermal Amplification,簡(jiǎn)稱(chēng)SLP-LAMP)。 本試驗(yàn)以單增李斯特菌hlyA基因?yàn)榘谢?應(yīng)用在線(xiàn)引物設(shè)計(jì)軟件(https://primerexplorer.jp/lamp4.0.0/index.html)設(shè)計(jì)篩選出一套具有一定莖環(huán)結(jié)構(gòu)的LAMP引物,建立了莖環(huán)型引物-環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)檢測(cè)單增李斯特菌的方法。對(duì)反應(yīng)時(shí)間、反應(yīng)溫度、Mg2+濃度等擴(kuò)增條件進(jìn)行優(yōu)化,確定25μL的SLP-LAMP反應(yīng)體系為:內(nèi)引物FIP和BIP各1.0μmol/L,外引物F3和B3各0.2μmol/L,dNTPs 0.4 mmol/L, MgCl2 4 mmol/L,10×Bst DNA聚合酶反應(yīng)緩沖液2.5μL,8 U/μL的Bst DNA聚合酶0.6μL, DNA模板1.5μL,滅菌的去離子水補(bǔ)足體系。SLP-LAMP反應(yīng)過(guò)程是首先在63℃水浴鍋中反應(yīng)50 min,然后將其放入80℃水浴鍋中,水浴5 min終止反應(yīng),通過(guò)肉眼觀察有無(wú)白色焦磷酸鎂沉淀,即可判斷是否發(fā)生了SLP-LAMP反應(yīng),亦可通過(guò)瓊脂糖凝膠電泳證實(shí)反應(yīng)是否發(fā)生。通過(guò)限制性?xún)?nèi)切酶ClaⅠ酶切擴(kuò)增產(chǎn)物,測(cè)得酶切片段與理論預(yù)期值相符,驗(yàn)證了方法的正確性。本研究對(duì)3株單增李斯特菌、5株李斯特屬的非單增李斯特菌株和14株其他食源性致病菌進(jìn)行特異性試驗(yàn)。結(jié)果表明,3株單增李斯特菌結(jié)果為陽(yáng)性,其他19株菌株為陰性。研究了6種從人工污染單增李斯特菌的奶粉中提取模板DNA的方法,結(jié)果表明SLP-LAMP檢測(cè)對(duì)制備模板DNA方法的要求不高。 本研究對(duì)SLP-LAMP法快速檢測(cè)單增李斯特菌進(jìn)行了研究,確定了檢測(cè)純培養(yǎng)物的靈敏度,人工污染檢出限,并與普通PCR檢測(cè)方法進(jìn)行了對(duì)比。結(jié)果表明:SLP-LAMP檢測(cè)單增李斯特菌的靈敏度為2.45×101 CFU/mL,其對(duì)人工污染奶粉樣品的檢出限為7.32×101 CFU/mL。采用試劑盒提取DNA,從樣品處理到報(bào)告結(jié)果,耗時(shí)2 h。而對(duì)照,PCR檢測(cè)單增李斯特菌純培養(yǎng)物的靈敏度為2.45×10~3 CFU/mL,人工污染單增李斯特菌的檢出限為7.32×10~3 CFU/mL。采用同樣方法提取DNA,從樣品處理到報(bào)告結(jié)果,耗時(shí)4 h。 研究表明,本研究建立的SLP-LAMP法檢測(cè)食品中單增李斯特菌的方法具有特異性強(qiáng)、靈敏度高,方便快捷、成本低等特點(diǎn),并且通過(guò)大量的試驗(yàn)證明,篩選設(shè)計(jì)帶有莖環(huán)型引物對(duì)避免反應(yīng)過(guò)程中出現(xiàn)假陽(yáng)性起到了一定的作用。為L(zhǎng)AMP的檢測(cè)提供了新的發(fā)展方向,該檢測(cè)方法操作簡(jiǎn)單,不需要昂貴的儀器設(shè)備、繁瑣的電泳過(guò)程,快速省時(shí),更利于基層檢驗(yàn)檢疫機(jī)構(gòu)的應(yīng)用。
[Abstract]:Listeria monocytogenes (LM), called Listeria monocytogenes (LM), is a kind of food borne pathogenic bacteria that can cause zoonosis. It belongs to the genus Lester bacteria. It is widely distributed in nature. It has been eaten by single Lester contaminated food, which can cause septicemia, abortion, meningitis and so on. The mortality rate is up to 30%. ~70% is one of the most important foodborne pathogens of human being. The detection, identification and control technology of single increasing Lester bacteria have been paid great attention. The method of detection has always used the traditional methods of separation and identification, which can not meet the requirements of modern rapid detection.
Loop-mediated isothermal amplification (LAMP) is a novel amplification technique of nucleic acid in vitro. It has the characteristics of simple, rapid, sensitive and specific. It has been applied to the detection of pathogenic bacteria and many other fields. The maximum local limit of this method is the easy to appear false positive. This study uses LAMP technology to examine The LAMP primers were designed, selected and improved, and the primers with a certain stem ring structure were selected to avoid the false positive of multiple primers under the same condition and possible complementary amplification, which could further improve the accuracy of LAMP detection, which could be called the stem ring primer -LAMP (Stemloop-Primer Loop-mediated Isoth). Ermal Amplification, short for SLP-LAMP).
In this experiment, a set of LAMP primers with a certain stem ring structure were designed and screened using the hlyA gene of single increasing Lester bacteria as the target gene, and the method of detecting monocytogenes by the stem ring primer ring mediated isothermal amplification technique was established. The reaction time and reaction time were determined. The conditions of temperature, Mg2+ concentration and other amplification conditions were optimized, and the SLP-LAMP reaction system of 25 mu L was determined as: the primers FIP and BIP each 1 mol/L, the outer primers F3 and B3 0.2 micron each, dNTPs 0.4 mmol/L, MgCl2 4, 2.5 micron polymerase reaction buffer of 10, 1.5 micron, 1.5 micron of the template, and the sterilized deionized water. The system.SLP-LAMP reaction process is first to react 50 min in a 63 centigrade bath pot, then put it in a 80 C water bath, and the water bath 5 min terminates the reaction. By the naked eye, there is no white magnesium phosphate precipitation, can determine whether the SLP-LAMP reaction has occurred, and the reaction can be confirmed by agarose gel electrophoresis. The enzyme Cla I enzyme was amplified and the enzyme cut fragments were measured in accordance with the theoretical expectation, and the correctness of the method was verified. This study conducted specific tests on 3 strains of Lester bacteria, 5 non single Lester strains of Lester and 14 other foodborne pathogens. The results showed that 3 strains of Lester bacteria were positive, and the other 19 strains were positive. 6 methods of extracting template DNA from the milk powder of Lester bacteria by artificial contamination were studied. The results showed that the SLP-LAMP test was not very demanding for the preparation of the template DNA method.
In this study, the rapid detection of monocyculia monocytogenes by SLP-LAMP method was studied. The sensitivity of the pure culture, the detection limit of artificial contamination and the comparison with the common PCR detection method were determined. The results showed that the sensitivity of SLP-LAMP detected by SLP-LAMP was 2.45 x 101 CFU /mL, and the detection limit for the samples of artificial contaminated milk was 7.3. The 2 x 101 CFU/mL. used the kit to extract DNA, from the sample to the report result, the time consuming 2 h. and the control. The sensitivity of the pure culture of the single increasing Lester bacteria by PCR was 2.45 x 10~3 CFU/mL, the detection limit of the Artificially Polluted single Lester bacteria was 7.32 * 10~3 CFU/mL. using the same method to extract DNA, from the sample to the report result, and the time consuming 4 h..
The study shows that the method of SLP-LAMP method for detecting Lester bacteria in food has the characteristics of specificity, sensitivity, convenience, low cost and so on. And through a large number of experiments, it is proved that the selection of the primers with stem ring primers plays a certain role in avoiding the false positive in the reaction process. For the detection of LAMP For the new direction of development, the detection method is simple to operate, does not need expensive instruments and equipment, complicated electrophoresis process, fast and time-saving, and is more conducive to the application of basic inspection and quarantine organizations.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：TS252.7;R155.5
本文編號(hào)：2121453
[Abstract]:Listeria monocytogenes (LM), called Listeria monocytogenes (LM), is a kind of food borne pathogenic bacteria that can cause zoonosis. It belongs to the genus Lester bacteria. It is widely distributed in nature. It has been eaten by single Lester contaminated food, which can cause septicemia, abortion, meningitis and so on. The mortality rate is up to 30%. ~70% is one of the most important foodborne pathogens of human being. The detection, identification and control technology of single increasing Lester bacteria have been paid great attention. The method of detection has always used the traditional methods of separation and identification, which can not meet the requirements of modern rapid detection.
Loop-mediated isothermal amplification (LAMP) is a novel amplification technique of nucleic acid in vitro. It has the characteristics of simple, rapid, sensitive and specific. It has been applied to the detection of pathogenic bacteria and many other fields. The maximum local limit of this method is the easy to appear false positive. This study uses LAMP technology to examine The LAMP primers were designed, selected and improved, and the primers with a certain stem ring structure were selected to avoid the false positive of multiple primers under the same condition and possible complementary amplification, which could further improve the accuracy of LAMP detection, which could be called the stem ring primer -LAMP (Stemloop-Primer Loop-mediated Isoth). Ermal Amplification, short for SLP-LAMP).
In this experiment, a set of LAMP primers with a certain stem ring structure were designed and screened using the hlyA gene of single increasing Lester bacteria as the target gene, and the method of detecting monocytogenes by the stem ring primer ring mediated isothermal amplification technique was established. The reaction time and reaction time were determined. The conditions of temperature, Mg2+ concentration and other amplification conditions were optimized, and the SLP-LAMP reaction system of 25 mu L was determined as: the primers FIP and BIP each 1 mol/L, the outer primers F3 and B3 0.2 micron each, dNTPs 0.4 mmol/L, MgCl2 4, 2.5 micron polymerase reaction buffer of 10, 1.5 micron, 1.5 micron of the template, and the sterilized deionized water. The system.SLP-LAMP reaction process is first to react 50 min in a 63 centigrade bath pot, then put it in a 80 C water bath, and the water bath 5 min terminates the reaction. By the naked eye, there is no white magnesium phosphate precipitation, can determine whether the SLP-LAMP reaction has occurred, and the reaction can be confirmed by agarose gel electrophoresis. The enzyme Cla I enzyme was amplified and the enzyme cut fragments were measured in accordance with the theoretical expectation, and the correctness of the method was verified. This study conducted specific tests on 3 strains of Lester bacteria, 5 non single Lester strains of Lester and 14 other foodborne pathogens. The results showed that 3 strains of Lester bacteria were positive, and the other 19 strains were positive. 6 methods of extracting template DNA from the milk powder of Lester bacteria by artificial contamination were studied. The results showed that the SLP-LAMP test was not very demanding for the preparation of the template DNA method.
In this study, the rapid detection of monocyculia monocytogenes by SLP-LAMP method was studied. The sensitivity of the pure culture, the detection limit of artificial contamination and the comparison with the common PCR detection method were determined. The results showed that the sensitivity of SLP-LAMP detected by SLP-LAMP was 2.45 x 101 CFU /mL, and the detection limit for the samples of artificial contaminated milk was 7.3. The 2 x 101 CFU/mL. used the kit to extract DNA, from the sample to the report result, the time consuming 2 h. and the control. The sensitivity of the pure culture of the single increasing Lester bacteria by PCR was 2.45 x 10~3 CFU/mL, the detection limit of the Artificially Polluted single Lester bacteria was 7.32 * 10~3 CFU/mL. using the same method to extract DNA, from the sample to the report result, and the time consuming 4 h..
The study shows that the method of SLP-LAMP method for detecting Lester bacteria in food has the characteristics of specificity, sensitivity, convenience, low cost and so on. And through a large number of experiments, it is proved that the selection of the primers with stem ring primers plays a certain role in avoiding the false positive in the reaction process. For the detection of LAMP For the new direction of development, the detection method is simple to operate, does not need expensive instruments and equipment, complicated electrophoresis process, fast and time-saving, and is more conducive to the application of basic inspection and quarantine organizations.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：TS252.7;R155.5
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