CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV

Hepatitis B remains a major global public health challenge, with particularly high prevalence in medically disadvantaged western Pacific and African regions. Although clinically available technologies for the qPCR detection of HBV are well established, research on point-of-care testing has not progressed substantially. The development of a rapid, accurate point-of-care test is essential for the prevention and control of hepatitis B in medically disadvantaged rural areas. The development of the CRISPR/Cas system in nucleic acid detection has allowed for pathogen point-of-care detection. Here, we developed a rapid and accurate point-of-care assay for HBV based on LAMP-Cas12a. It innovatively solves the problem of point-of-care testing in 10 min, particularly the problem of sample nucleic acid extraction. Based on LAMP-Cas12a, visualization of the assay results is presented by both a fluorescent readout and by lateral flow test strips. The lateral flow test strip technology can achieve results visible to the naked eye, while fluorescence readout can achieve real-time high-sensitivity detection. The fluorescent readout-based Cas12a assay can achieve HBV detection with a limit of detection of 1 copy/μL within 13 min, while the lateral flow test strip technique only takes 20 min. In the evaluation of 73 clinical samples, the sensitivity and specificity of both the fluorescence readout and lateral flow test strip method were 100%, and the results of the assay were fully comparable to qPCR. The LAMP-Cas12a-based HBV assay relies on minimal equipment to provide rapid, accurate test results and low costs, providing significant practical value for point-of-care HBV detection.     

基于协同过滤推荐的学生社团组织平台设计与实现

当代大学生的校园生活非常丰富，有各种各样的组织及社团，大部分学生都参加了一个以上的社团组织。但目前各高校的社团组织工作繁琐，管理非常低效，且各自为政，信息孤立。针对这些问题，开发了Org社团组织协作平台，通过线上线下结合，给不同的用户设立相应的权限，提供消息发布、活动签到、物品租赁、日程安排、社区交流等功能。平台基于Android Studio进行开发，后台使用Linux、Apache、MySql、PHP组合。     

基于LAMP架构开发Web应用的优势

LAMP（Linux＋Apache＋Mysql＋PHP/Perl）是一种web网络应用和开发环境,是四种开源软件技术的组合。采用LAMP技术的主要特点是：性能稳定、数据安全、运行速度超快、不涉及版权问题。LAMP是一种使用相对简单、方便．但功能强大的应用服务平台,广泛应用于网站建设、办公自动化、电子政务等Web应用系统。     

LAMP技术在微生物检测中的应用

LAMP(1oop-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。     

Endpoint Visual Detection of Three Genetically Modified Rice Events by Loop-Mediated Isothermal Amplification

Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%–0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops.     

副溶血弧菌tdh基因LAMP检测技术的建立

环等温扩增技术(Loop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异性、高效、快速地扩增靶基因的DNA扩增技术。根据副溶血弧菌特异性的毒力基因tdh保守序列,本文设计1套特异性引物对该基因进行环等温扩增,同时对反应条件进行优化,建立携带tdh基因的致病性副溶血弧菌的LAMP快速检测技术。结果表明,LAMP最适反应在60.8℃恒温、45min内完成,与其它常见的细菌无交叉反应。LAMP方法的细菌DNA最低检出限为35.5fg/反应管,灵敏度较PCR方法高10倍。对扇贝样品中分离的菌株的检测表明,LAMP方法对检测致病性副溶血弧菌具有良好的可靠性。     

Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

ABSTRACT: Loop-mediated isothermal amplification (LAMP) is a novel molecular detection method that is more rapid and simpler than PCR. Products can be detected by turbidity using one temperature without the need for expensive PCR equipment. Our objective was to sensitively detect Salmonella Typhimurium from pork products within 1 d using the LAMP assay. Pork chop and pork sausage samples (25 g) were inoculated with high (10⁸ to 10⁶ CFU) and low (10⁵ to 10⁰ CFU) inocula of S. Typhimurium. Serial dilutions in phosphate buffered saline were plated on XLT4 agar either immediately or after selective preenrichment in tetrathionate broth (225 mL) for 10-h at 37 °C. Nucleic acid was extracted using the TRIzol^® method from 1-mL samples. The LAMP assay using 6 specific invA gene primers and Bst DNA polymerase reaction mix was carried out at 62 °C for 90 min in a waterbath. Turbid products were detected visually and by agarose gel electrophoresis. Improved Salmonella detection at 10² CFU/25 g for both pork chop and sausage was obtained after 10-h enrichment and 10⁶ CFU/25 g without enrichment for both products. This assay can detect Salmonella from pork within 1 d, significantly faster than traditional methods that take >5 d. This method shows tremendous potential for routine diagnostics and monitoring of Salmonella by the pork industry. Practical Application: The novel loop-mediated isothermal amplification (LAMP) assay is a rapid, specific, and sensitive method that has potential application for routine diagnostics of Salmonella from pork products. The isothermal method does not require expensive equipment such as a PCR thermocylcer but only a simple waterbath for amplification within 90 min. Detection is even simpler by visual eye or turbidimeters that are less expensive than fluorescent spectrophotometers or real-time PCR machines. All these advantages make it a practical approach for routine use by processing industries to rapidly detect Salmonella in their environment and to implement appropriate control strategies. To improve detection sensitivities, preenrichment followed by selective enrichment may be necessary. Even so, the entire assay can be completed at the most within two 8-h working shifts.     

应用LAMP方法检测动物源性食品中沙门菌

目的运用两种方法对动物源性食品中沙门菌进行检测,找出一种更加快速、敏感、特异、简便的检验方法。方法采集60份市售的禽、畜等生肉及60份奶制品,分别采用LAMP方法和国家标准方法GB/T4789.4—2008对沙门菌进行分离与鉴定。结果 120份采集样品中,LAMP方法检出2例,阳性率1.67%;国家标准方法检出1例,阳性率0.83%。LAMP方法的符合率为99.2%,敏感性为100%,特异性为99.2%。结论 LAMP检测法快速、特异、简便,该方法与国标法的阳性率差异无显著性。     

基于LAMP＋AJAX素质拓展系统的开发应用

通过大学生素质拓展网上认证系统,教育管理者可以利用信息技术手段,极大地提高教育管理工作的效率。针对大学生素质拓展网上认证系统开发及应用,介绍LAMP技术与AJAX技术结合的新特点,着重讨论如何运用LAMP和AJAX技术相结合实现网上认证系统所设计的功能,给出系统的设计及实现方案。教育管理者通过使用该套网上认证系统可以进一步整合日常教学主渠道以外有助于提高学生综合素质的各种资源,引导和帮助广大学生有计划地完善智能结构,实现全面成长成才。