环介导等温扩增检测猪血中金黄色葡萄球菌

采用环介导等温扩增(LAMP)技术,检测猪血中金黄色葡萄球菌.以金黄色葡萄球菌(CMCC10201)的femA基因作为靶序列,设计LAMP和PCR引物,通过凝胶电泳,判断检测结果.对8株常见致病菌进行LAMP特异性实验,表明对金黄色葡萄球菌的检测具有很高的特异性;LAMP检测金黄色葡萄球菌的灵敏度为8.7CFU/mL,直接检测猪血中金黄色葡萄球菌的检出限为8.7×102CFU/mL,PCR法的检出限为8.7×103CFU/mL.本实验所建立的快速检测猪血中金黄色葡萄球菌的LAMP法具有较高的特异性和敏感性,能够满足快速检测的需要.     

多重LAMP—熔解曲线法检测食品中两种食源性致病菌

针对沙门氏菌的侵袭蛋白基因(inv A)和金黄色葡萄球菌的耐热核酸酶基因(nuc)设计LAMP引物,优化反应条件,并分别对反应产物进行熔解曲线分析,判断扩增结果,从而建立食品中沙门氏菌和金黄色葡萄球菌多重LAMP—熔解曲线检测方法。对9株目标菌和23株非目标菌的检测未出现假阳性和假阴性结果,并可通过熔解曲线的熔解温度分析确定所含目标菌;对产物的测序分析表明所得序列与目的基因序列吻合;对目标菌的检测灵敏度均可达到10 fg/μL;对232份样品的检测结果显示,其中2份生猪肉中检出金黄色葡萄球菌。该方法具有良好的检测特异性,并可为食源性致病菌的快速检测提供一种重要技术手段。     

基于Linux流媒体的开放教学、实践平台的搭建

随着网络技术的发展,高效的教学实践平台对高校教育影响巨大。建立基于Linux流媒体的开放教学、实践平台对共享教学资源、学生自主学习基于流媒体的教学内容,学习网站管理和Web数据库编程,提高实际的编程能力都有实际意义。通过该平台学生可以在课后与教师交流、答疑,并获得预习和复习的指导。本文阐述了建立普通高校规范、统一、开放的教学实践平台的思想,并且讲述了如何利用开源软件Linux、LAMP、vsFtP和real Server架设基于Linux流媒体的开放教学、实践平台。     

基于PHP的在线请假系统的设计与实现

通过对系统功能模块进行需求分析及设计．采用LAMP技术进行学生在线请假管理系统的开发。本系统可实现教师对学生请假信息的在线管理，促进高校学生工作管理的信息化．方便服务于广大师生。     

Rapid detection of Listeria monocytogenes in raw milk with loop-mediated isothermal amplification and chemosensor

Loop-mediated isothermal amplification (LAMP) allows a rapid amplification of nucleic acids under isothermal conditions. It can be combined with a rhodamine-based dual chemosensor for much more efficient, field-friendly detection of Listeria monocytogenes. In this report, LAMP was performed at 63 °C for 10 min, followed by a rapid reaction of DNA amplification and the byproduct, pyrophosphate ion, with a rhodamine-based dual chemosensor and Cu(2+) is visualized as a disappearance of red color. The detection limit of L. monocytogenes by the LAMP-chemosensor was 8 to 10 cells per reaction tube, and the total assay time including 10 min for rapid DNA extraction was approximately 30 min. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with the ISO 10560 reference method. The results showed that the LAMP-chemosensor method has the advantages of better sensitivity and speed and less dependence on equipment than the standard Polymerase Chain Reaction for specifically detecting low levels of L. monocytogenes DNA, and this can be useful in the field as a routine diagnostic tool. PRACTICAL APPLICATION: The LAMP-chemosensor method reported here provided a powerful tool for detection of L. monocytogenes in raw milk samples due to its specificity, sensitivity, and rapidity.     

Rapid Detection of Salmonella enterica Subspecies enterica serovar Typhimurium by Loop Mediated Isothermal Amplification (LAMP) Test From Field Chicken Meat Samples

Considering the importance of Salmonella enterica subsp. enterica serovar Typhimurium in the foodborne diseases, a Typhimurium specific loop mediated isothermal amplification (LAMP) test was standardized for its rapid detection in chicken meat. The Optimum results were obtained at 64^oC and 70 min temperature-time combination. The sensitivity of LAMP and PCR were compared with serial 10-fold dilution of the 100 ng of DNA. The LAMP test detected 2 pg DNA per reaction tube, whereas PCR detected 200 pg DNA per reaction. Therefore, the LAMP test was considered 100 times more sensitive than the PCR. The specificity of LAMP and PCR analyzed with six different isolates of non-Salmonella and 22 serovars of non-Typhimurium. None of these isolates were found positive by both LAMP and PCR. Twenty-eight pure isolates of Salmonella Typhimurium from diverse sources were also examined by Typhimurium specific LAMP and were all found positive. Two-hundred twenty-five field chicken meat samples were screened by cultural, PCR, and LAMP methods. The LAMP and PCR tests were performed by using DNA isolated from 8 h and 18 h enrichment samples, respectively. Typhimurium specific LAMP was shown to be in 100% correlated with cultural and PCR methods. However, LAMP test delivered the results within 26 h without sophisticated equipment while PCR and cultural methods took 48 h and 6 d, respectively. The LAMP test developed in this study has potential to detect and as well as differentiate S. Typhimurium from other Salmonella serovars.     

Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of Schistosoma mansoni and Strongyloides spp. -A Laboratory Approach to Point-Of-Care

Loop-mediated isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-time fluorescence detection using fluorophore-labelled probes attached to oligonucleotide sequences complementary to the target nucleic acid. This methodology allows the simultaneous detection of multiple targets (multiplexing) in one LAMP assay. A method for multiplexing LAMP is the amplification by release of quenching (DARQ) technique by using a 5′-quencher modified LAMP primer annealed to 3′-fluorophore-labelled acting as detection oligonucleotide. The main application of multiplex LAMP is the rapid and accurate diagnosis of infectious diseases, allowing differentiation of co-infecting pathogens in a single reaction. Schistosomiasis, caused among other species by Schistosoma mansoni and strongyloidiasis, caused by Strongyloides stercoralis, are the most common helminth-parasite infections worldwide with overlapping distribution areas and high possibility of coinfections in the human population. It would be of great interest to develop a duplex LAMP to detect both pathogens in the same reaction. In this study, we investigate the use of our two previously developed and well-stablished LAMP assays for S. mansoni and Strongyloides spp. DNA detection in a new duplex real-time eight-primer system based on a modified DARQ probe method that can be performed in a portable isothermal fluorimeter with minimal laboratory resources. We also applied a strategy to stabilize the duplexed DARQ-LAMP mixtures at room temperature for use as ready-to-use formats facilitating analysis in field settings as point-of-care diagnostics for schistosomiasis and strongyloidiasis.     

环介导恒温扩增技术可视化检测携带tdh基因的致病性副溶血性弧菌

建立了环介导恒温扩增技术检测携带tdh基因的致病性副溶血性弧菌。基于副溶血性弧菌高度保守的tdh基因序列,设计了6条特异性引物,两条外引物F3、B3,两条内引物FIP、BIP及两条环引物LF、LB。在Bst DNA Polymerase作用下,60℃恒温水浴进行扩增。对10种细菌共21株菌进行LAMP扩增,所试6株副溶血性弧菌均为阳性,说明引物具有高度特异性。本LAMP方法对纯培养物的灵敏度可达到9.42cfu/m L。对污染食品中副溶血性弧菌的灵敏度为25.3cfu/25g,40~60min内即可完成检测。本方法操作简便、特异性强、灵敏度高,可以为临床提供简单、快速、高灵敏度和高特异性的检测应用。      

一个可自定义个性化参数的项目流程管理系统

论文基于LAMP架构构建网站服务器,采用ThinkPHP框架设计了一个用户可定制个性化参数的通用项目流程管理系统.该系统不仅包括通常项目流程管理系统中的核心功能模块,而且还提供给用户自定义个性化参数的功能.实验结果表明,不同需求的用户可灵活地定制管理对象及数据,该系统将据此自动生成其用户专用的项目流程管理系统.     

环介导等温扩增技术快速检测沙门菌

环介导等温扩增(loop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异、高效、快速地扩增靶序列的DNA扩增新技术.以沙门菌(Salmonella spp.)为研究对象,根据其特异性的invA基因,设计了一套特异性引物对该基因进行了LAMP,同时优化了其反应条件,建立了沙门菌的LAMP快速检测技术.结果表明,LAMP的最佳反应条件为外引物浓度5 pmol/L、内引物浓度40pmol/L,Mg2 浓度6mmol/L,dNTP浓度0.8mmol/L,甜菜碱浓度0.8mmol/L,Bst DNA聚合酶8u,反应温度63℃,反应时间1 h.在此条件下,LAMP检测沙门菌DNA的敏感度达10fg/反应,且与其他常见的细菌无交叉反应.其对牛奶样品的检出量为102cfu/mL,适合于食品中污染沙门菌的快速检测.