【作者】 杨红；

【导师】 张伟；

【作者基本信息】 河北农业大学 ， 农产品加工及贮藏工程， 2011， 硕士

【摘要】 金黄色葡萄球菌(Staphylococcus aureus)简称金葡菌,是食品中常见的污染菌,该菌认为是引起细菌性食物中毒最普遍的原因之一。国内外由金葡菌引发的食物中毒屡屡发生,金葡菌所引起的食物中毒已日益成为全球性的公共卫生问题,严重危害人类安全与健康。因而,应该建立一种方便快捷的检测金葡菌的方法是食品安全问题中的一项重要的举措,可以起到防患于未然。目前针对金葡菌的检测方法主要有传统检测方法,免疫学方法和PCR方法。传统检测方法操作繁琐,检测时间长,且灵敏度较低;免疫学方法的特异性和灵敏度也不是很理想,PCR方法敏感、快速,但是由于需要昂贵的仪器设备、繁琐的电泳过程,而使其难以在基层普及和推广。环介导等温扩增方法(LAMP)是一种由日本科学家Notomi开发的新型的核酸扩增技术,可以扩增特异性核酸序列,具有高特异性、高灵敏性,简便快速和成本低的特点,已在诸多领域广泛应用。本试验是采用环介导等温扩增技术快速检测食品中金葡菌。以金葡菌的高度保守序列-耐热核酸酶nuc序列为靶基因,运用在线引物设计软件(https://primerexplorer.jp/lamp3.0.0/index.html)设计LAMP引物。对镁离子浓度、dNTPs浓度、反应时间等扩增条件进行优化,确定25μL的LAMP反应体系为:内引物(FIP和BIP)各1.6 mmol/L,外引物(F3和B3)各0.4 mmol/L,环引物(LB和LF)各0.8 mmol/L,4.0 mmol/L dNTPs,3 mmol/L MgSO4,10×Bst DNA聚合酶反应缓冲液,8 U的Bst DNA聚合酶大片段,2.5μL DNA模板,并用灭菌双蒸水补足体系。LAMP的反应过程为:先在64℃水浴锅中反应20 min,然后在80℃水浴锅中水浴10 min终止反应。反应结束后,通过肉眼观察有无白色焦磷酸镁沉淀即可判断LAMP反应是否发生,亦可通过琼脂糖凝胶电泳证实反应是否发生。为了比较LAMP检测金葡菌的灵敏度和人工污染检出限,以PCR方法作对照。PCR反应的引物是LAMP反应的两条外引物(F3和B3)。结果表明,LAMP检测金葡菌的灵敏度为1.25 CFU/反应管(2.5×10~1CFU/mL),人工污食品的检出限为10.3 CFU/反应管(2.06×10~2CFU/mL)。PCR检测金葡菌的灵敏度为1.25×10~2 CFU/反应管( 2.5×10~3CFU/mL ),人工污染食品的检出限为1.03×10~3 CFU/反应管(2.06×10~4CFU/mL)。采用试剂盒法提取DNA,从样品处理到报告结果,LAMP方法耗时1 h,PCR方法耗时3 h。利用LAMP方法对40株肠道致病菌进行特异性试验。结果表明,24株金葡菌为阳性,其它16株非金葡菌为阴性。初步研究了8种模板制备方法对LAMP检测食品中金葡菌的影响。结果表明,LAMP方法对制备模板DNA的要求不高。通过对实际样品进行检测,将本方法与国家标准GB/T4789.10-2010方法进行比较,确定LAMP方法敏感性为100%,特异性为97.93%,符合率为98.5%。研究表明,本研究建立的LAMP法检测食品中金葡菌的方法具有特异性强、灵敏度高,方便快捷、成本低等特点,为金葡菌的检测提供了新的发展方向,并且由于该检测方法有操作简单,不需要昂贵的仪器设备、繁琐的电泳过程,快速省时等优点,更利于一些基层检验检疫机构的应用, LAMP法有望成为简易的常规检测手段,对于提高食品卫生水平、保证食品安全和推动食品国际贸易发展具有重要的意义。更多还原

【Abstract】 Staphylococcus aureus （S. aureus） is one of the most common and important pathogenic bacterium. In the word, there are many cases that are cause by S. aureus. Alimentary toxicosis caused by S. aureus is now a very serious problem worldwide. To solve this problem, a fast and highly efficient method for detecting S. aureus should be established.Current standard detection methods require lengthy culture enrichment steps to increase target bacterial numbers before isolation and identification can be performed. These conventional food microbiological techniques often require several days to detect bacterial pathogens present in food at low levels.The sensitivity and specificity of immunological Methods are unsatisfactory. Recently, a number of new molecular biological methods for rapid detection of bacteria have been reported, including polymerase chain reaction （PCR）. The PCR methods provide powerful tools for rapid, specific, and sensitive detection of food-borne pathogens and are considered reliable alternatives for traditional bacteriological methods. However, owing to the expensive systems required and complicate electrophoretic analysis, this application is still not very common in laboratories.Loop-mediated isothermal amplification （LAMP） is a new microbiological method devised by the Japanese scientist Notomi. Because of it can provide a sensitive , specific, simple and cost-effective test in detecting microbes and viruses。The loop-mediated isothermal amplification method （LAMP） had been used frequently in a lot of areas including pathogens’repid detection.We used the the heat-stable nuclease （nuc） gene of S. aureus in the GenBank database （GenBank accession V01281.1）,which was the high specificity and conserved sequence, as target sequences and used Primer Explorer software, version 3（http://primerexplorer.jp/lamp3.0.0/index.html）, to design LAMP primers. The reaction conditions were optimized including temperature,time and Mg²⁺ concentration etc. The LAMP mixture was made in 25μL of reaction mixture containing a 1.6μM concentration of each inner primer （FIP and BIP）, a 0.4μM concentration of each outer primer （F3 and B3）, a 0.8μM concentration of the loop primer（LB and LF）, 4.0μM each deoxynucleoside triphosphate, 3.0μM MgSO₄,10×Bst DNA polymerase reaction buffer, 8 U of the Bst DNA polymerase large fragment, 2.5μL isolated DNA templates and sterilized double-distilled water. The mixture was incubated at 64℃for 20 min and then heated to 80℃for 10 min to terminate the reaction. We can obtain the results of detection when the white precipitate was observed by visual examination.For an assessment of the sensitivity and detection limit of artificial contamination of LAMP, PCR with the two outer primers （F3 and B3） of LAMP with two loop primers was performed. We extracted DNA using the same DNA kit method. The detection limit of pure bacterial culture was 1.25 CFU per reaction tube（2.5×10¹CFU/mL）and the detection limit of artificially contaminated food sample was 10.3 CFU per reaction tube（2.06×10²CFU/mL） with LAMP detection for two hours. In contrast, the detection limit of pure bacterial culture was 1.25×10² CFU per reaction tub（e2.5×10³CFU/mL）and the detection limit of artificially contaminated food sample was 1.03×10³ CFU per reaction tube（2.06×10⁴CFU/mL）with PCR detection for three hours. Our results showed this LAMP assay had a high specificity for the detection of Staphylococcus aureus by amplifying a fragment of nuc gene of one Staphylococcus aureus strain rather than other sixteen non- S. aureus strains.The effects of eight methods of DNA extraction from S. aureus in food sample on detetion of S. aureus were compared. The results showed that LAMP had fewer requirements for performing a successful detection. Sensitivity of the method in S. aureus detection was 100%, with 97.93% specificity and a 98.5% match ratio when compare using Chinese National standard GB/T4789.10-2010. The result indicated that the sensitivity of detection of S. aureus by LAMP was superior to those of other methods. Its specificity was also excellent when distinguishing S. aureus from other bacteria. Finally, the sensitivity, specificity, and matched route of LAMP were comparable to other methods. Given that LAMP is cheaper, more convenient, and faster, it will be readily accepted by many quarantine institutes requiring microbiological detection methods. The LAMP method is an easy and convenient testing method for improving food sanitation, maintaining food safety, as well as developing international trade.更多还原