The detection of Atlantic cod (Gadus morhua) using loop mediated isothermal amplification in conjunction with a simplified DNA extraction process

Atlantic cod (Gadus morhua) is a commercially important species of white fish, and one of three species legally identifiable as cod in the UK. Mislabelling of G. morhua does occur, as does the substitution of G. morhua for less expensive species. Sensitive molecular tests based on PCR have been developed for this species, but they have limitations, including the need for expensive thermal cycling equipment, and complex DNA extraction procedures. A loop mediated isothermal amplification (LAMP) assay was designed for the G. morhua cytochrome b gene, which was capable of detecting 0.1% w/w G. morhua in a homogenised raw fish mix. The LAMP assay was also able to detect G. morhua DNA when a rapid sample preparation was used, involving heating 100 mg of fish in a 1 ml aliquot of water and testing the supernatant, showing a higher tolerance of amplification inhibitors than a PCR assay. The LAMP assay did not generate a positive result when challenged with a range of non-target species, including Gadus macrocephalus, and Gadus chalcogrammus, indicating a high level of specificity. Direct detection of a positive reaction using propidium iodide was also demonstrated.     

Assessment of the Campylobacter jejuni and C. coli in broiler chicken ceca by conventional culture and loop-mediated isothermal amplification method

In a two-year survey of the 24 Japanese broiler chicken flocks at 9 farms from 2013 to 2014, C. jejuni/C. coli prevalence was assessed in a total of 131 slaughtered broiler chicken cecal samples by conventional culture methods and loop-mediated isothermal amplification (LAMP) assay. While 93 samples were C. jejuni/C. coli-negative, 38 (29.0%) showed Campylobacter loads of between 6.4 and 9.0 log CFU/g of ceca in conventional culture methods. The performance of LAMP assay was 100% accurate in terms of diagnostic sensitivity (38/38), specificity (93/93). Furthermore, LAMP assay enabled direct screening of C. jejuni and C. coli in cecal samples from broiler chicken chickens as rapid and cost-effective detection within 90 min and less than 1 US dollar, which can help monitor release of Campylobacter-contaminated chicken into the food chain, thereby reducing the incidence and public health risk of campylobacteriosis. Seasonal changes in C. jejuni and C. coli prevalence in broiler chicken ceca were significantly correlated with the frequency of food poisoning incidents caused by these bacteria in Japan.     

Detection and quantification of pathogenic Vibrio parahaemolyticus in shellfish by using multiplex PCR and loop-mediated isothermal amplification assay

Vibrio parahaemolyticus is a halophilic bacterium that commonly inhabits the marine and estuarine environments. This organism is also one of the leading causative pathogen of gastroenteritis often related to consumption of raw or undercooked seafood. In this study, molluscan shellfish (bloody clams and surf clams) and crustaceans (shrimps) were monitored in wet markets and hypermarkets. Two molecular methods were employed and compared to detect total and pathogenic V. parahaemolyticus in MPN enrichments: multiplex PCR and LAMP assay. The multiplex PCR was optimized to detect the total (toxR+), tdh+ and trh+ V. parahaemolyticus. On the other hand, the LAMP assay was employed to target the pathogenic strains only, the tdh+ and trh+, respectively. Out of 232 samples examined, 229 (98.7%) were positive for V. parahaemolyticus with counts ranging from 30 to >110, 000 MPN/g. Positive samples for tdh+ V. parahaemolyticus were obtained in 77 out of 232 (33.1%) samples ranging from 30 to >110, 000 MPN/g. Meanwhile, positive samples for trh+ were identified in 16 out of 232 (6.9%) samples examined ranging from 30 to 9600 MPN/g. Detection of samples with presence of tdh+ genes did not vary between methods, but a significant difference was observed when the LAMP assay was compared to PCR to detect trh+ V. parahaemolyticus. Therefore, on occasions where the density of the targeted genes is low, the LAMP assay serves as a better alternative. Nonetheless, this study constitutes an assessment of presence of total and potentially pathogenic V. parahaemolyticus in shellfishes for domestic consumption revealing the potential risk of contracting vibriosis if precautions and safety measures are not properly managed.     

环介导等温扩增技术检测食品中酸土环脂芽孢杆菌

目的：利用环介导等温扩增技术建立食品中酸土环脂芽孢杆菌快速检测方法。方法：针对酸土环脂芽孢杆菌16S序列设计特异引物,再优选反应体系,用显色法检测实验结果。结果：该方法能够在63 ℃条件下1 h内检出食品中酸土环脂芽孢杆菌,所设计的引物有良好的特异性;灵敏度达6.7 CFU/mL（弱阳性）。结论：该方法具有高效、特异性强和敏感性高等特点,可满足酸土环脂芽孢杆菌快速检测筛选的要求。     

食品中牛奶过敏原成分LAMP检测方法的建立

目的 建立食品过敏原牛奶成分LAMP检测方法,并与实时荧光PCR(real-time PCR)检测方法比对。方法 针对牛线粒体细胞色素b(cyt-b)基因设计LAMP引物并建立反应体系,在特异性和灵敏度方面与real-time PCR检测方法比对。结果 本研究建立的LAMP方法检测9份不同品牌的牛奶和羊奶及其加工制品,没有出现交叉反应,具有良好的特异性。通过添加试验方法的检测灵敏度为0.5 %,与real-time PCR方法检测灵敏度相当。检测了69份实际样品,检测结果与real-time PCR检测结果一致。结论 本研究建立的食品过敏原牛奶成分LAMP检测方法简单经济,检测结果可靠,可有效缩短检测时间,适用于过敏原牛奶成分的检测,具有良好的应用前景。     

环介导恒温扩增法检测肉制品中致病微生物

目的:建立一种食品中微生物核酸快速检测技术,在此基础上开发的简便、快速、灵敏,不依赖仪器适合基层应用的核酸快速检测试剂盒;并探索该试剂盒在肉及肉制品微生物检测中的应用。方法:根据单核细胞增生李斯特氏菌特有的靶序列vir R基因设计引物,采用环介导恒温扩增技术构建检测体系,优化、验证检测的特异度、检出限等指标,并与传统分离培养法比较。结果:本研究中的检测方法及试剂盒能专一的检出肉制品中的单增李斯特菌,对其他近源菌株检测呈阴性结果,检出限达1.81×103CFU/mL,与传统方法具有相同的灵敏度;检测操作简便快速,极少的设备依赖,适合规模推广。     

Real-time loop-mediated isothermal amplification for the CaMV-35S promoter as a screening method for genetically modified organisms

A method has been developed to detect genetically modified organisms (GMOs). The detection method is based on the loop-mediated isothermal amplification (LAMP) reaction. Cauliflower mosaic virus 35S (CaMV-35S) promoter gene, a widespread genetic element, was amplified by a set of LAMP primers. One of the characteristics of the LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of byproduct, pyrophosphate ion, is produced, yielding a white precipitate of magnesium pyrophosphate in the LAMP reaction mixture. The measurement of the turbidity of the reaction mixture allows easy detection of amplification of CaMV-35S promoter gene without the need for gel electrophoresis. With this new technique we analyzed Roundup Ready soybean, and found GMO content ranging from 0.5% to 5%.     

基于LAMP的网络渗透测试系统设计与实现

为了更好的完成对目标系统的远程安全评估,设计实现了一种基于LAMP架构的网络渗透测试系统。该系统实现功能包括:基于B/S模式的系统Web框架,运用NASL攻击脚本插件实现的漏洞扫描模块,基于攻击图的渗透方案自动生成模块,统一调用的多数据库通用引擎。测试结果表明,系统能够有效针对目标主机完成信息探测、漏洞扫描、渗透攻击、测试评估等功能,系统性能稳定,操作简单,可扩展性强。     

一种低成本高质量的协作学习平台的研究

针对国内现有协作学习平台在开销与学习质量上的不足,设计出了一种适合中小企业或高校信息建设的协作学习平台框架。通过低廉的成本来实现较高质量的协作学习服务,其中低成本主要从组建、发布、维护等几方面来体现,高质量分别从学习任务,学习环境、学习评价等多方面论述,最终构建出了能真正解决中小企业在信息化学习建设支出方面的难题,也能实现对学习者高效率的培养的协作学习平台。