环介导等温扩增法快速检测耐甲氧西林金黄色葡萄球菌

建立一种应用环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法快速检测耐甲氧西林金黄色葡萄球菌(methicillin-resistan Staphylococcus aureus,MRSA)中mecA和spa基因的检测方法。以金葡菌和其他相关菌种为试验对象,分别用聚合酶链式反应(ploymerase chain reaction,PCR)和LAMP方法检测mecA和spa基因。结果表明：LAMP法在64℃等温条件下可在60min内成功扩增基因,且与传统PCR方法结果相同;通过琼脂糖凝胶电泳可知,LAMP对mecA和spa基因的检测限分别为每菌管102个和10个细胞;肉眼可检测到的mecA和spa基因的检测限分别为每菌管103个和10个细胞;然后用LAMP法检测人工污染原料乳样本中MRSA,用LAMP法检测原料乳中的mecA和spa基因与PCR方法显示出相同的结果。LAMP方法可快速检测mecA和spa基因,此方法可应用于原料乳中MRSA的检测。     

Evaluation of a loop-mediated isothermal amplification (LAMP) method for rapid on-site detection of horse meat

Detection of horse DNA by loop-mediated isothermal amplification (LAMP) seems one of the most promising methods to meet the criteria of fast, robust, cost efficient, specific, and sensitive on-site detection. In the present study an assessment of the specificity and sensitivity of the LAMP horse screening assay was made and outcomes were compared with the EURL-AP (European Union Reference laboratory for Animal Proteins in feeding stuffs) qPCR method. The specificity was tested with DNA samples from seven other species. The sensitivity of the LAMP assay was subsequently challenged with different percentages of horse DNA in cattle DNA and different percentages of horse meat in cattle meat. Both qPCR and LAMP were able to reliably detect horse DNA or meat below 0.1%, but LAMP was able to do so in less than 30 min. The DNA of other species did not result in a response in the LAMP horse assay. These features show that the LAMP method is fast, specific, and sensitive. Next, 69 processed meat samples were screened for the presence of horse DNA. The results showed that the LAMP horse assay, combined with a simple and fast on-site DNA extraction method, results in similar outcomes as the EURL-AP qPCR method and is thus a promising screening assay to be used outside the laboratory. Only samples that are screened on-site as suspect in the LAMP horse assay, need to be brought to the laboratory for confirmation with the more laborious EURL-AP qPCR reference method.     

新冠病毒的现场可视化LAMP检测及其冷链食品分析应用

建立了一种基于环介导等温扩增技术(Loop-mediated Isothermal Amplification,LAMP)和核酸分子"光开关"[Ru(phen)₂dppz]²⁺相结合的新型冠状病毒高灵敏可视化检测方法。分别采用保温杯代替实验室恒温装置,采用蓝光手电代替蓝光透射仪,实现了新冠病毒的现场快速可视化LAMP检测。针对新型冠状病毒N基因设计特异性序列引物,采用[Ru(phen)₂dppz]²⁺分子光开关的特性检测LAMP扩增产物,结果可以借助蓝光手电直接通过肉眼观察是否出现红色荧光进行判定。该方法可检测到单个拷贝数的新型冠状病毒基因片段,并具有高度特异性,检验快速,整个过程仅需40min即可目视观察结果。对新冠病毒假病毒人工污染的模拟冷链食品进行检测,结果与目前新型冠状病毒检测金标准实时荧光定量PCR法吻合度为100%。该方法仅需保温杯和蓝光手电,可以作为实时荧光PCR方法的补充,为食品新冠病毒的现场筛查提供快速高效的技术支持。     

水产品和水体中创伤弧菌现场可视化环介导恒温扩增快检方法的建立及应用

目的 基于环介导恒温扩增(LAMP)技术建立水产品和养殖水域中创伤弧菌现场可视化快速、简便的检测方法。方法 以创伤弧菌作为研究对象,分别采用煮沸法和离心柱法提取基因组DNA,优化简便、快速的弧菌DNA提取方法;以创伤弧菌gyrB基因作为靶基因,设计筛选最佳的适于LAMP的引物序列,分别通过LAMP荧光扩增曲线(加SYTO-9 荧光染料),以及LAMP可视化观察扩增产物的颜色变化(加钙黄绿素),开展特异性、灵敏度和重复性试验分析。结果 确定煮沸法为适合于弧菌基因组DNA提取的快捷方法;优化筛选的引物可以特异地检测创伤弧菌,检测核酸浓度的灵敏度可以达到10 fg/μL,并且结果稳定、可靠。采用该方法进行实际样品的检测应用,在558份水体样品和655份水产品样品中,创伤弧菌阳性检出率分别为9.01%和8.60%,阳性检出率高于传统的分离培养鉴定结果。结论 低技术要求、低设备成本以及检测时间短使得可视化LAMP快检方法成为现场可使用的一个理想选择,对于水产养殖业来说也更方便。     

基于LAMP的网站流量分析系统图表功能的设计与实现

阐述了基于LAMP架构开发设计的网站流量分析系统的结构模型,并对JpGraph配置进行了详细的分析,对图表功能做了进一步的实现,展示了运行效果。     

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.