Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65 °C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis.     

Anti-inflammatory property of n-hexadecanoic acid: structural evidence and kinetic assessment

Ester bond hydrolysis of membrane phospholipids by Phospholipase A₂ and consequent release of fatty acids are the initiating steps of inflammation. It is proposed in this study that the inhibition of phospholipase A₂ is one of the ways to control inflammation. Investigations are carried out to identify the mode of inhibition of phospholipase A₂ by the n‐hexadecanoic acid. It may help in designing of specific inhibitors of phospholipase A₂ as anti‐inflammatory agents. The enzyme kinetics study proved that n‐hexadecanoic acid inhibits phospholipase A₂ in a competitive manner. It was identified from the crystal structure at 2.5 Å resolution that the position of n‐hexadecanoic acid is in the active site of the phospholipase A₂. The binding constant and binding energy have also been calculated using Isothermal Titration Calorimetry. Also, the binding energy of n‐hexadecanoic acid to phospholipase A₂ was calculated by in silico method and compared with known inhibitors. It may be concluded from the structural and kinetics studies that the fatty acid, n‐hexadecanoic acid, is an inhibitor of phospholipase A₂, hence, an anti‐inflammatory compound. The inferences from the present study validate the rigorous use of medicated oils rich in n‐hexadecanoic acid for the treatment of rheumatic symptoms in the traditional medical system of India, Ayurveda.     

Evaluation of percutaneous absorption of the repellent diethyltoluamide and the sunscreen ethylhexyl p‐methoxycinnamate‐loaded solid lipid nanoparticles: an in‐vitro study

Objectives Diethyltoluamide and ethylhexyl p‐methoxycinnamate (OMC) are two active ingredients in insect repellent and sunscreen products, respectively. The concurrent application of these two substances often increases their systemic absorption, compromising the safety and efficiency of the cosmetic product. In this study, diethyltoluamide and OMC were incorporated into solid lipid nanoparticles, a colloidal drug delivery system, to reduce percutaneous absorption and avoid toxic effects and also maintain the efficacy of the two active compounds on the skin surface for a long duration. Methods Solid lipid nanoparticles were prepared based on an ultrasonication technique and characterized by differential scanning calorimetry (DSC) analyses. In‐vitro studies determined the percutaneous absorption of diethyltoluamide and OMC. Key findings DSC data carried out on unloaded and diethyltoluamide‐ and/or OMC‐loaded solid lipid nanoparticles highlighted that diethyltoluamide and OMC modified the temperature and the enthalpy change associated to the calorimetric peak of solid lipid nanoparticles. The concurrent presence of the two compounds in the solid lipid nanoparticles caused a synergic effect, indicating that the lipid matrix of nanoparticles guaranteed a high encapsulation of both diethyltoluamide and OMC. Results from the in‐vitro study demonstrated that the particles were able to reduce the skin permeation of the two cosmetic ingredients in comparison with an oil‐in‐water emulsion. Conclusions This study has provided supplementary evidence as to the potential of lipid nanoparticles as carriers for topical administration of cosmetic active compounds.     

The kinetics of thermal instability in nanocrystalline silver and the effect of heat treatment on the antibacterial activity of nanocrystalline silver dressings

The kinetics of nanocrystalline silver dressing heat treatment was investigated via isothermal heat treatments at 90 °C, 100 °C, and 110 °C lasting 2–50 h. Bactericidal efficacy of the dressings was measured via log reductions, while bacteriostatic longevity was determined via plate-to-plate transfer corrected zones of inhibition. Morphological evolution of the dressing was studied by X-ray diffraction, scanning electron microscopy, and X-ray photoelectron spectroscopy, while changes in heat flow were measured by differential scanning calorimetry. Increasing temperature increased the rate at which dressing bactericidal activity and bacteriostatic longevity decreased. Once changes in dressing properties began, they occurred nonlinearly with time. The earliest biological, chemical, and physical indicators of altered dressing properties were loss of bacteriostatic longevity, silver–oxygen bonds, and fine features, respectively. An early change in heat flow appeared to be responsible for these indicators, while a later change corresponded to rapid grain growth occurring after a critical crystallite size (30 nm) was reached. The grain growth exponent was determined to be 2.8 for temperatures of 100–110 °C, with an activation energy of 177 kJ/mol, suggesting that normal grain growth occurred, with volume and/or grain boundary diffusion as the dominant forms of diffusion. The thermal instability of nanocrystalline silver should be accounted for during production, storage, and use of dressings. The properties required for nanosilver antimicrobial efficacy demonstrated in this study, as well as its thermal instability, should be taken into consideration for the development of nanosilver products in the future.     

应用环介导等温扩增法快速检测O139群霍乱弧菌

目的 建立一种适合基层检验部门及小型实验室使用的快速检测O139群霍乱弧菌的方法.方法 应用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),针对O139霍乱弧菌wbfR基因设计4条引物(2条内引物、2条外引物);优化LAMP反应条件和反应体系,对反应体系中的引物、dNTP、Mg2+/Mn2+离子及Calcium等浓度进行优化;并对13株种系背景明确的霍乱弧菌不同实验对照株、30株O139群霍乱弧菌地方分离株、10株O1群霍乱弧菌地方分离株、32株其他肠道菌进行检测,验证该方法的特异性;通过肉眼目测或电泳检测比较结果.结果 所有O139群霍乱弧菌经LAMP榆测均呈绿色并电泳有阶梯状条带为阳性,O1群霍乱弧菌及其他肠道菌均检测呈橙色并电泳无相应条带为阴性;该体系最低检测限为63 CFU/反应;检测结果在白光下通过肉眼即可判断;从菌株核酸的提取至检测完成仅需1.5 h左右.结论 本研究建立的LAMP方法能够快速、灵敏、特异地检测O139群霍乱弧菌,无需昂贵的仪器,简单方便,非常适合基层检验部门或小型实验室以及流行病学人员于应急车上或现场监测等使用,值得推广.     

A physical approach to modify the hydraulic reactivity of α-tricalcium phosphate powder

A microsized α-tricalcium phosphate (α-TCP) powder was calcined at various temperatures (350 °C < T < 800 °C) for various durations (1–24 h) and the resulting physico-chemical and reactivity changes were measured. Without calcination, the α-TCP powder started reacting within minutes after contacting a 0.2 M Na₂HPO₄ solution as measured by isothermal calorimetry. The overall reaction was finished within a few days. After calcination at 350 °C  T  550 °C for 24 h, no significant changes in the crystalline composition, crystallite size, particle size or specific surface area were noticed. However, the powder reactivity was progressively changed. More specifically, the hydraulic reaction of the powders calcined at 500 and 550 °C only started after 2–3 h whereas the overall hydraulic reaction was only slightly postponed, suggesting that physical or chemical changes had occurred at the particle surface. As mainly physical changes were detected at the particle surface during calcination at 500 °C, it was speculated that the appearance of this reaction delay (= induction time) was due to the disappearance of surface defects during the calcination step, i.e. to the need to create surface defects to induce dissolution and hence reaction.     

环介导等温扩增法鉴定检测冬虫夏草

目的建立冬虫夏草的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,实现对冬虫夏草的快速检测。方法针对冬虫夏草的CS2 serine protease(csp2)基因设计LAMP内外引物对;利用CTAB法提取冬虫夏草DNA;优化扩增反应条件;采用包括冬虫夏草在内的6种不同虫草进行LAMP扩增,并对阳性产物酶切鉴定以检测其特异性,通过对模板DNA的10倍梯度稀释检测其灵敏度;紫外灯下观察凝胶电泳或加入荧光染料SYBR Green I的扩增产物。结果设计的LAMP引物可以特异地针对冬虫夏草的csp2基因进行LAMP扩增,产物可被限制性内切酶Taq1酶切,且有较高的灵敏度,检出限达6 pg/mL。结论 LAMP法可以实现对冬虫夏草的快速鉴定检测,在中药鉴定中有着广阔的应用前景。     

Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF

The porins OmpF and OmpC are trimeric β-barrel proteins with narrow channels running through each monomer that exclude molecules > 600 Da while mediating the passive diffusion of small nutrients and metabolites across the Gram-negative outer membrane (OM). Here, we elucidate the mechanism by which an entire soluble protein domain (> 6 kDa) is delivered through the lumen of such porins. Following high-affinity binding to the vitamin B(12) receptor in Escherichia coli, the bacteriocin ColE9 recruits OmpF or OmpC using an 83-residue intrinsically unstructured translocation domain (IUTD) to deliver a 16-residue TolB-binding epitope (TBE) in the center of the IUTD to the periplasm where it triggers toxin entry. We demonstrate that the IUTD houses two OmpF-binding sites, OBS1 (residues 2-18) and OBS2 (residues 54-63), which flank the TBE and bind with K(d)s of 2 and 24 μM, respectively, at pH 6.5 and 25 oC. We show the two OBSs share the same binding site on OmpF and that the colicin must house at least one of them for antibiotic activity. Finally, we report the structure of the OmpF-OBS1 complex that shows the colicin bound within the porin lumen spanning the membrane bilayer. Our study explains how colicins exploit porins to deliver epitope signals to the bacterial periplasm and, more broadly, how the inherent flexibility and narrow cross-sectional area of an IUP domain can endow it with the ability to traverse a biological membrane via the constricted lumen of a β-barrel membrane protein.     

霍乱弧菌环介导等温扩增LAMP技术检测

目的 应用环介导等温扩增技术(LAMP)进行霍乱弧菌的快速检测.方法 针对霍乱弧菌的管家基因(mdh)设计4条特异性引物(2条内引物和2条外引物),建立快速检测食品中霍乱弧菌的环介导等温扩增方法;应用该方法对17种细菌共42株菌进行LAMP扩增,并进行确证,全面评估该方法的灵敏度、特异性、准确性等.结果 该方法检测21株霍乱弧菌均得到阳性扩增结果,其余21株非霍乱弧菌均未扩增出条带,扩增反应最佳反应时间为60min,反应温度为65℃.霍乱弧菌基因组DNA和纯培养物的检测灵敏度分别约为40 fg和50 cfu/mL.对模拟食品样品进行直接检测,检测限为70 cfu/g.结论 该方法具有特异性强、灵敏度高,操作简便快速、不需要复杂仪器设备,适用于食品的快速检测.