FTA滤膜与环介导等温扩增技术结合快速检测消毒乳中的蜡样芽孢杆菌

目的:建立环介导等温扩增技术(LAMP)快速检测蜡样芽孢杆菌的方法.方法:根据公布的蜡样芽孢杆菌溶血素基因的hblA基因的保守序列设计引物,将FTA滤膜提取DNA与LAMP法相结合,检测蜡样芽孢杆菌和人工污染蜡样芽孢杆菌的消毒乳.结果:LAMP方法检测灵敏度高,蜡样芽孢杆菌的检出限为4.4 cfu/mL,比PCR检测灵敏度高10倍;人工污染消毒乳中蜡样芽孢杆菌的检出限为57 cfu/mL.LAMP扩增可在20 min内完成,对消毒乳中蜡样芽孢杆菌的检测(包括DNA提取、LAMP和电泳)可在2 h内完成.结论:初步建立LAMP检测蜡样芽孢杆菌的快速检测方法.该方法灵敏度高、特异性好、耗时短.为食品中蜡样芽孢杆菌快速检测构建了一个技术平台.     

Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae,V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products

Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872–1:2007 and TS 21872–2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.     

环介导恒温扩增法(LAMP)检测金黄色葡萄球菌

建立一种能够快速准确地检测金黄色葡萄球菌的LAMP方法。根据金黄色葡萄球菌的femA基因设计了引物,然后进行LAMP反应条件的优化、特异性和灵敏度的检测并与实际样品进行检出率的比较。LAMP方法特异性好,最佳反应温度为61℃,只对金黄色葡萄球菌进行扩增;灵敏度高,金黄色葡萄球菌的检测灵敏度为8～9cfu/mL时仍能检出。LAMP方法检测金黄色葡萄球菌特异性强、灵敏度高、时间短且操作简便,有望成为快速检测金黄色葡萄球菌的新方法。     

环介导等温扩增技术快速检测产气荚膜梭菌的研究

目的:建立环介导等温扩增技术(LAMP)快速检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌(ATCC13124)的α毒素全基因序列为保守序列,设计内、外、环引物,肉眼观察白色沉淀并判断检测结果。结果:LAMP检测产气荚膜梭菌的灵敏度为2.92×102CFU/mL,人工污染产气荚膜梭菌的全脂乳的检测限为15.7CFU/mL,耗时仅1h。对照PCR检测产气荚膜梭菌的灵敏度为2.92×105CFU/mL,人工污染产气荚膜梭菌的全脂乳的检出限1.57×105CFU/mL。采用同样的方法提取DNA,耗时3h。结论:建立LAMP快速、特异检测产气荚膜梭菌的方法,该方法灵敏度是PCR的1000倍,为食品中产气荚膜梭菌的检测构建了一个技术平台。     

食品中大肠杆菌O157:H7的几种检测方法的比较

目的评估平板分离培养法、免疫磁珠分离(IMS)法、VIDAS全自动酶标免疫测试系统、BAX全自动病原菌检测系统及环介导等温扩增(LAMP)技术在食品中检验肠出血型大肠埃希菌O157∶H7的特异性、敏感性。方法使用平板分离培养法、免疫磁珠分离法、VIDAS法、BAX法及LAMP法对人工制备的染菌猪肉样本进行检测,并对这几种方法进行比较。结果 BAX法和LAMP法的检出率最高,分别是89.1%和85.9%,免疫磁珠法和VIDAS法检出率次之,分别是75.0%和78.1%,传统分离培养法为43.8%。结论 BAX法和LAMP法具有快速、高效、特异性好、敏感性高的特点,可快速筛选食品中可能存在的肠出血型大肠埃希菌O157∶H7。     

实时浊度LAMP法检测豆制品中转基因成分

通过设计转基因大豆内源基因(Lectin)、外源基因NOS和2个转基因大豆品系(GTS 40-3-2,MON89788)特异性LAMP引物,建立快速检测转基因大豆以及鉴定转基因大豆品系的LAMP体系,并选取国内豆制品利用CTAB法进行DNA提取,并针对上述基因进行PCR和LAMP扩增的对比,建立起了检测大豆制品中转基因成分的定性的LAMP方法。结果表明:4个LAMP方法均有较好的特异性,除了NOS、GTS 40-3-2的检测限为0.1%外,其他LAMP方法的灵敏度为0.01%,均能满足实际检测要求;在实时浊度LAMP法检测大豆制品转基因成分的鉴定中,传统的CTAB法能成功提取大豆粗加工制品的DNA,且DNA纯度能够进行核酸扩增反应。利用转基因外引物进行的普通PCR结果与实时浊度LAMP结果是一致的,说明建立的转基因实时浊度LAMP检测方法有很好的准确性。     

食品中单增李斯特菌环介导等温扩增检测技术

目的：建立一种快速、简单地检测食品中单增李斯特菌的环介导恒温基因扩增方法(loop-mediated isothermal amplification,LAMP),并初步建立荧光定量LAMP检测单增李斯特菌的方法。方法：根据LAMP方法的原理,设计LAMP检测引物,建立LAMP检测方法,同时对方法的特异性、灵敏度、重复性及初始拷贝数的对数值与反应时间之间的线性关系进行评估。结果：使用LAMP引物可以在0.5h内完成检测工作;LAMP检测技术的灵敏度高出聚合酶链式反应检测技术100倍以上,检出限达到1.72×101拷贝/反应,与其他食源性致病菌无交叉反应,平均实验间变异系数小于5%;反应时间与初始模板浓度的常用对数值有良好的线性关系(R2=0.9994)。结论：本方法具有快速、灵敏、特异、操作简单、设备要求低的特点,具有广阔的应用前景。     

Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling

Electron microscopy (EM) is traditionally employed as a follow‐up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on‐section fluorescence labelling with on‐section immunogold labelling. In addition to antibody labelling of LAMP‐1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane‐associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane‐associated antigens.     

LAMP技术在食品致病菌检测中的研究进展

环介导的恒温扩增(LAMP)是近年来发展起来的一种新的恒温核酸扩增技术,该技术具有特异性强、敏感性高、反应快、设备简单等特点,已被应用于病原菌诊断、食品致病菌检测等领域的研究。本文重点阐述了近年来国内外学者应用LAMP技术在食品致病菌检测中的研究进展。     

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.