Loop-mediated isothermal amplification-based microfluidic chip for pathogen detection

Abstract

Due to the significant growth of food production, the potential likelihood of food contamination is increasing. Foodborne illness caused by bacterial pathogens has considerably increased over the past decades, while at the same time, the species of harmful microorganisms also varied. Conventional bacterial culturing methods have been unable to satisfy the growing requirement for food safety inspections and food quality assurance. Therefore, rapid and simple detection methods are urgently needed. The loop-mediated isothermal amplification (LAMP) technology is a highly promising approach for the rapid and sensitive detection of pathogens, which allows nucleic acid amplification under isothermal conditions. The integration of the LAMP assay onto a microfluidic chip is highly compatible with point-of-care or resource-limited settings, as it offers the capability to perform experiments in combination with high screening efficiency. Here, we provide an overview of recent advances in LAMP-based microfluidic chip technology for detecting pathogens, based on real-time or endpoint determination mechanisms. We also discuss the promoting aspects of using the LAMP technique in a microfluidic platform, to supply a guideline for further molecular diagnosis and genetic analysis.     

阻尼器响应放大技术研究与应用进展

目前，阻尼器在工程结构减隔震控制领域的应用已极为广泛，但存在中小地震作用下阻尼器的位移和速度等较小时，阻尼器难以充分发挥耗能能力的问题。因此近年来阻尼器响应放大技术得到了国内外学者的广泛重视，并利用连杆机构、齿轮机构、杠杆机构、跨层支撑等提出了多种阻尼器响应放大装置。全面综述国内外阻尼器响应放大技术的研究和应用进展，包括装置构造和作用机理等。同时，分析现有阻尼器响应放大技术在遭遇极罕遇地震作用时阻尼器更容易失效等方面的不足，指出响应放大技术的研究和发展方向，以期为阻尼器响应放大技术的发展和极罕遇地震作用下确保工程结构的安全提供有效思路。     

基于碳纳米管信号放大的卡那霉素高灵敏分析方法的建立

卡那霉素是一种氨基糖苷类抗生素,具有广谱抗菌性,在动物医疗中应用广泛,从而导致了其在动物源性食品中广泛残留,因此检测食品中卡那霉素的残留很有必要。作者以多壁碳纳米管(Multiwalled Carbon Nanotubes,MWCNTs)作为载体,通过EDC/NHS方法偶联抗体与信号分子辣根过氧化物酶(Horseradish Peroxidase,HRP),合成多酶复合颗粒MWCNTs-Abs-HRP;建立基于MWCNTs-Abs-HRP多酶复合颗粒的检测方法,相比于已建立的传统竞争ELISA,灵敏度提高约5倍;并成功将此方法应用于牛奶样本中卡那霉素的检测。     

Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.     

PCR-DGGE技术检测速冻饺子馅料中微生物区系

以速冻饺子为研究对象,对速冻饺子冻前的微生物多样性进行研究分析,为速冻饺子馅料的存放期限及防腐技术提供参考,避免后期出现卫生问题。利用传统分离培养的方法,对饺子肉馅中的细菌进行分离,得到30株纯菌株。通过菌株形态学鉴定、基因组DNA提取、聚合酶链式反应(polymerase chain reaction,PCR)扩增及16S rDNA测序及序列比对分析来鉴定菌株的属种、构建种群结构进化树。结果表明:30株纯菌株中有14个属,22个种,优势菌分布在γ-变形菌纲中的假单胞菌属。     

鸟嘌呤四链体DNAzyme在微生物、生物小分子和核酸检测中的研究进展

鸟嘌呤四链体脱氧核酶(deoxyribozyme,DNAzyme)是一种具有类过氧化物酶催化活性的脱氧核酶,广泛应用于微生物和核酸的分析与检测中。鸟嘌呤四链体DNAzyme在H_2O_2存在时可以催化氧化ABTS、TMB等底物,结合PCR、等温扩增技术以及生物传感器等多种核酸靶标信号放大技术实现待测靶标的便捷灵敏的可视化检测,因此受到了国内外研究者们的高度关注。优化鸟嘌呤四链体的序列和环境因素,能够提高该DNAzyme的催化活性,有助于提高可视化检测方法的灵敏度。本文主要对鸟嘌呤四链体DNAzyme的结构与酶学性质及其应用于微生物、生物毒素和疾病标志物等的基于核酸的检测策略进行综述,为进一步推动鸟嘌呤四链体DNAzyme的广泛应用提供理论基础和技术支持。     

A simple and sensitive aptasensor with rolling circle amplification for viable Cronobacter sakazakii detection in powdered infant formula

Cronobacter sakazakii is a foodborne, emerging opportunistic pathogen that causes severe bacteremia, necrotizing enterocolitis, and sepsis with a mortality rate of up to 80%. In this study, we developed a simple and sensitive fluorescent turn-off aptasensor with rolling circle amplification assay for viable C. sakazakii detection in powdered infant formula. The results showed that the proposed aptasensor has good performance and specificity for detecting viable C. sakazakii in pure culture and powdered infant formula samples within 3 h. Under the optimal reaction conditions, there is a linear relationship between fluorescent intensity at 490 nm and logarithmic concentration of C. sakazakii in the range of 2.7 × 10⁵ to 2.7 × 10² cfu/mL, with a limit of detection of 2.7 × 10² cfu/mL in pure culture. The proposed aptasensor achieved a recovery of 104 to 111% in pure culture, and 96 to 107% in spiked powdered infant formula samples. The proposed aptasensor does not require complicated DNA extraction steps or antibodies, and can be performed at 37°C, making it a convenient and sensitive strategy for C. sakazakii detection.