Loop‐mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp

Aims

Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop‐mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite.

Methods and Results

A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA‐labelled nanogold probe, followed by salt‐induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0·02 fg total DNA) than a conventional nested PCR (0·2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP‐AuNP assay was specific for E. hepatopenaei.

Conclusions

Without sacrificing sensitivity or specificity, the new LAMP‐AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp.

Significance and Impact of the study

The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.     

应用多种教学方法,建设活跃的生物化学课程

生物化学授课过程中,教师通过丰富课堂资源、进行问题导向式教学、加强在线学习、建立多元化的课程考核方式及设定定时答疑制度等多种方法,激发学生学习兴趣,提升生物化学教学和学习效果,建设"笑起来""动起来"的活跃的课程。同学们普遍认为,生物化学课程内容充实有趣,教学形式多样,并且在课堂上学到了很多前沿知识。     

硕士研究生参与本科生组织学与胚胎学实验课教学的体会

硕士研究生通过参与本科生组织学与胚胎学实验课教学,可加深对本专业知识的理解,建立更合理的知识结构,完善自身的思维体系,提高综合能力,从中发现自身不足之处并积极弥补。硕士研究生参与本科生教学具有独特的优势,对提升其综合素质有所助益。     

基于固态基底的SERS免疫检测新方法研究

目的:通过引入新型表面增强拉曼散射(SERS)检测探针(Au-DTNB-Tyr NPs)和金标银染技术,建立基于固态硅片基底的SERS免疫检测新技术。方法:羊抗人IgM-HRP作为检测抗体,在硅片基底上检测不同浓度的人IgM,HRP催化SERS检测探针沉积,利用金标银染技术增强SERS信号。结果:所建立的SERS免疫检测新方法检测人IgM的检测限为10 pg/mL,且SERS信号强度与人IgM浓度具有良好的线性关系(R2=0.993)。结论:基于硅片基底的SERS免疫检测新技术可高灵敏地定量检测人IgM,为实现固态硅片基底对多种抗原的高通量集成化检测奠定了基础。     

以“环境微生物学”为载体的环境生态工程专业本科生科创能力培养

环境生态工程是一个设立较晚的新专业,其基本的教学体系和专业课程设计还有待摸索和完善。"环境微生物学"作为环境生态工程专业普遍设置的专业必修课,其授课内容和形式如何适应专业整体的培养需求是一个值得探讨的问题。我们结合所在学校、学院的特点和优势,提出将融入海洋特色和生态学思想的"环境微生物学"作为载体,结合课上理论教学、学生课堂报告、课下专题培训和实验技能培养等形式,培养环境生态工程专业本科生的科创能力。这可为同专业开设"环境微生物学"课程的教师提供教学参考,也可以为其他院校环境生态工程专业本科生科创能力的培养提供借鉴。     

全英文“环境工程微生物学”的国家线下一流本科课程成长之路

国家一流本科课程的评审认定是教育部全面深化教育教学改革的重要举措,也是提升本科教学质量的重要一环,极大地促进了任课教师对标评价量规进行教研教改。为促进环境工程专业核心基础课的教改,“环境工程微生物学”全英课程组在先进的教育思想、方法和教育心理学的指导下,对教学理念、课程内容、教学组织和实施等多方面进行了大胆的改革和创新,注重课程思政和因材施教,增加课堂教学的师生互动和生生互动;针对工科类学生的培养目标,引入实际工程的应用案例,在课内外补充环境微生物工程领域研究的最新进展和教师的科研成果,注重提升课程的高阶性、创新性和挑战度,从多方面强化知识、能力、素质的有机融合,学生学习效果明显提高。申报并获认定为首批国家线下一流本科课程和广东省一流本科课程。     

The Developmental Origins of Osteoporosis

Osteoporosis is one of the most prevalent skeletal disorders and has enormous public health consequences due to the morbidity and mortality of the resulting fractures. This article discusses the developmental origins of osteoporosis and outlines some of the modifiable and non-modifiable risk factors in both intrauterine and postnatal life that contribute to the later onset of osteoporosis. Evidence for the effects of birth size and early growth in both preterm and term born infants are discussed and the role of epigenetics within the programming hypothesis is highlighted. This review provides compelling evidence for the developmental origins of osteoporosis and highlights the importance of osteoporosis prevention at all stages of the life course.     

A new spectrofluorimetric method for the determination of some tetracyclines based on their interfering effect on resonance fluorescence energy transfer

A rapid, simple, inexpensive and highly sensitive spectrofluorimetric method was developed for the determination of trace amounts of some tetracyclines (TCs), namely tetracycline hydrochloride (TCH), oxytetracycline hydrochloride (OTCH) and minocycline hydrochloride (MCH). Binding rhodamine B (RhB) to gold nanoparticles (Au NPs) resulted in quenching of the fluorescence of RhB by a resonance energy transfer (FRET) mechanism, with Au NPs as the energy acceptors. The presence of TCs caused the release of RhB molecules and recovered their fluorescence, and this was used as a basis for the quantitative determination of TCs. The reaction was monitored spectrofluorimetrically by measuring the increase in fluorescence of RhB at 572 nm starting 5 min after mixing the reagents in Tris buffer solution (pH 6.5). The effect of various experimental factors such as buffer type, pH, concentrations of the involved reagents and reaction time were studied to optimize the reaction conditions. Under optimum conditions, the calibration graphs were linear within the ranges 2.08 × 10^?9–1.04 × 10^?6 mol/L, 2.01 × 10^?9–1.00 × 10^?6 mol/L and 2.02 × 10^?9–1.01 × 10^?6 mol/L and detection limits (LODs) of 0.61 × 10^?9, 0.32 × 10^?9 and 0.66 × 10^?9 mol/L were calculated for TCH, OTCH and MCH, respectively, with corresponding percent relative standard deviations (%RSDs) of 1.18, 1.21 and 1.54 (n = 5). The method was successfully applied to the determination of TCs in drinking water, human urine, bovine milk and breast milk samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Utility of gold nanoparticles in luminescence determination of trovafloxacin: comparison of chemiluminescence and fluorescence detection

Two novel sensitive sequential injection chemiluminescence analysis and fluorescence methods for trovafloxacin mesylate detection have been developed. The methods were based on the enhancement effect of gold nanoparticles on luminol–ferricyanide–trovafloxacin and europium(III)–trovafloxacin complex systems. The optimum conditions for both detection methods were investigated. The chemiluminescence signal was emitted due to the enhanced effect of gold nanoparticles on the reaction of luminol–ferricyanide–trovafloxacin in an alkaline medium. The response was linear over a concentration range of 1.0 × 10^–9 to 1.0 × 10^–2 mol/L (%RSD = 1.3), (n = 9, r = 0.9991) with a detection limit of 1.7 × 10^–10 mol/L (S/N = 3). The weak fluorescence intensity signal of the oxidation complex of europium(III)–trovafloxacin was strongly enhanced by gold nanoparticles and detected at λ_ex = 330 and λ_em = 540 nm. Fluorescence detection enabled the determination of trovafloxacin mesylate over a linear range of 1.0 × 10^–8 to 1.0 × 10^–3 mol/L (%RSD = 1.2), (n = 6, r = 0.9993) with a detection limit of 3.3 × 10^–9 mol/L. The proposed methods were successfully applied to the determination of the studied drug in its bulk form and in pharmaceutical preparations. The results were treated statistically and compared with those obtained from other reported methods. Copyright © 2015 John Wiley & Sons, Ltd.     

胃癌血清的表面增强拉曼光谱研究

表面增强拉曼光谱技术在癌症诊断领域已经有了广泛的研究和应用,本文利用金纳米溶胶为增强基底,采用便携式近红外拉曼光谱系统,对89例胃癌患者及正常人血清样本进行了SERS光谱探测。结果表明,血清的特征峰主要归属于氨基酸,其次是核酸、糖类及脂类。相比于正常人血清SERS光谱,胃癌血清中归属于核酸的特征峰强度都较高,大部分归属于蛋白质的特征峰强度较低,与医学研究结论相符。说明SERS技术可以有效地反映胃癌患者和正常人血清的差异,为后期SERS技术快速诊断胃癌提供了实验基础。