PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state

Vibrio parahaemolyticus is a crucial foodborne pathogen. The viable but non-culturable (VBNC) state of V. parahaemolyticus cannot be detected by traditional culture methods. The objective of this study was to develop and evaluate a method that combines propidium monoazide (PMA) treatment with real-time fluorescent loop-mediated isothermal amplification (RF-LAMP) to detect and quantify VBNC cells of V. parahaemolyticus. Different states of cells were treated with PMA in dark for 5 min and subsequently exposed to a 650 W halogen lamp for 5 min. The cells were collected and DNA was amplified by RF-LAMP. The primers which targeted six distinct regions in the tlh gene of V. parahaemolyticus were used for the PMA-RF-LAMP assay. The results indicated that the treatment with 4 μg/mL of PMA and a further exposure to light for 5 min was suitable for PMA-RF-LAMP to distinguish viable cells from dead cells of V. parahaemolyticus. The developed assay exhibited remarkably high specificity, sensitivity and rapidity, without any cross reaction with the tested non-V. parahaemolyticus strains. There was good linear correlation between Ct values and log copy/mL in the range of 6.8–6.8 × 10⁷ copy/mL, with the reaction endpoints less than 30 cycles (30 min). It could detect as low as 14 copy/g of V. parahaemolyticus in spiked seafood samples (pomfret, shrimp, scallop, oyster and salted fish) without any interference from food matrices, dead cells and other bacteria.     

番茄溃疡病菌的环介导等温核酸扩增技术

目的 建立番茄溃疡病菌的环介导等温核酸扩增技术检测方法。方法 应用环介导等温核酸扩增技术(LAMP)对番茄溃疡病菌的16S rRNA特异性基因进行扩增, 采用4对引物识别目的片段的6个特异性区域, 62 ℃恒温1 h完成扩增。结果 设计的引物与对照菌皱纹假单胞菌、茄假单胞菌、丁香假单胞菌番茄致病变种都没有扩增反应, 表现了较好的特异性。这些对照菌在茄科蔬菜上易引起类似症状。结论 LAMP检测程序便捷, 所需设备简单, 其结果可以通过肉眼观察来判断, 比常规PCR灵敏且能更早得到反应结果, 因此该技术具有更大的优势。     

LAMP技术在微生物检测中的应用

LAMP（100p—mediated isothermal amplification）技术是一种新的恒温核酸扩增技术,具有特异性强、等温灵敏、操作简单、产物易检测等优点。该技术已被用于多种病原微生物的检测。本文综述了LAMP技术的原理,及其在几种微生物检测中的应用,并对其应用前景做了展望。     

在Ubuntu平台上部署基于Snort的入侵检测系统

Snort是一套免费的轻量级入侵检测系统,当今的很多入侵检测系统都以其为参照。虽然Snort软件体积非常小巧,但是其部署有一定的难度。为了以更直观的方式监测系统的运行状况,通常部署Snort系统时除了要安装Snort软件本身外,还需要大量的相关软件支持。文章介绍了在Ubuntu平台上架设snort系统的具体过程。系统以MysQL数据库存储入侵检测信息,用BASE工具显示检测结果。     

基于LAMP的高性能Web服务器的架构

文章提出了基于LAMP的高性能Web服务器的架构方案,采用了Apache日志、Webalizer日志分析、Cacti流量监控、入侵检测的方法,架构了一个完善的、稳定的、安全的、低廉的高性能Web服务器,满足了中小型企业的要求。     

基于开源LAMP架构的Web打印技术

通过对现有B/S架构打印方法进行分析对比,针对它们的缺陷提出了一种基于开源LAMP架构的跨平台、精确、有效的Web打印新技术,并详细介绍了其原理,最后给出了具体的实现方法.     

Compact optical diagnostic device for isothermal nucleic acids amplification

We recently reported the successful use of the loop-mediated isothermal amplification (LAMP) reaction for hepatitis B virus (HBV) DNA amplification and its optimal primer design method. In this study, we report the development of an integrated isothermal device for both amplification and detection of targeted HBV DNA. It has two major components, a disposable polymethyl methacrylate (PMMA) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of DNA amplification by-product, magnesium pyrophosphate. We have established a correlation curve (R² = 0.99) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. For the applications of rapid pathogens detection, we also have established a standard curve (R² = 0.96) by using LAMP reaction with a standard template in our device. Moreover, we also have successfully used the device on seven clinical serum specimens where HBV DNA levels have been confirmed by real-time PCR. The result indicates that different amounts of HBV DNA can be successfully detected by using this device within 1 h.     

嵌入式Web控制终端的物联网移动E-Laboratory平台

为了进一步提高大学校园设备资源的利用率,实现多种实验装置的网络化控制和用户终端移动化,基于物联网技术,将LAMP移植到采用ARM架构的微处理器上,通过搭建嵌入式Web控制终端,对Web服务平台进行了详细的设计,实现了多种实验装置的网络化控制;将单自由度机械臂系统这一实验装置与EWC_T连接,基于Android操作系统上开发了一个应用,设计并搭建了Mobile Experiment系统,实现了网络化实验室平台的用户终端的移动化;通过实验证明该系统能够可靠地完成对实验装置的远程控制,并具有低功耗、低成本和便携性好等优点。     

基于环介导等温扩增技术的产呕吐毒素 蜡样芽胞杆菌特异性检测

本研究旨在建立食品中产呕吐毒素蜡样芽胞杆菌的快速检测方法。基于产呕吐毒素蜡样芽胞杆菌合成酶基因cesB靶基因,设计4条特异性引物(2条内引物、2条外引物),建立环介导等温扩增技术(LAMP)。然后采用2株产呕吐毒素蜡样芽胞杆菌、19株蜡样芽胞杆菌和41株非蜡样芽胞杆菌验证了该LAMP具有很好的特异性。LAMP检测的灵敏度在DNA水平上达到1.49 pg/μL,纯菌的灵敏度为5×10~3 cfu/mL。人工污染米饭样品,当起始污染量为2 cfu/g时,37℃增菌培养6 h,用试剂盒法和煮沸法提取的DNA,都可以检出产呕吐毒素蜡样芽胞杆菌。采用此LAMP方法进行了72份实际样品的检测,检出2份产呕吐毒素蜡样芽胞杆菌阳性样品,与传统方法一致。因此,本研究建立的LAMP检测方法可应用于食品中产呕吐毒素蜡样芽胞杆菌的快速特异检测。     

Sensitive and rapid detection of Alicyclobacillus acidoterrestris using loop-mediated isothermal amplification

BACKGROUND: A loop‐mediated isothermal amplification (LAMP) assay was developed for the rapid detection (within 2 h) of Alicyclobacillus acidoterrestris. The assay detected the species‐specific DNA sequence of the 16S–23S rRNA internal transcribed spacer. RESULTS: The eight strains of A. acidoterrestris were successfully amplified, but six strains of other bacillus Acidocaldarius and 13 bacterial species other than bacillus Acidocaldarius were not. The sensitivity of the LAMP assay was at 4.50 × 10^?2 cfu per tube. This sensitivity is greater than that obtained by polymerase chain reaction (PCR) assay. The LAMP assay was examined further for its ability to detect A. acidoterrestris in juice samples. The results were compared with those of conventional PCR detection. CONCLUSION: Results indicate that the proposed LAMP assay is a rapid, specific and sensitive method for detecting A. acidoterrestris. As the amplification has been conducted under isothermal conditions, only a water bath or heating block is needed to maintain the required temperature. Thus, the method can be generalised and popularised easily in the future. Copyright © 2011 Society of Chemical Industry