Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing

The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification.     

Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification

We describe a novel technique for a simple, rapid, and reliable quantitative detection of specific DNA sequences using an alternately binding quenching probe (AB-QProbe) that binds to either the gene of interest (target) or an internal standard (competitor) in combination with loop-mediated isothermal amplification (LAMP). The AB-QProbe is a singly labeled oligonucleotide bearing a fluorescent dye at the 5' end. The fluorescence intensity of the AB-QProbe reflects the ratio of the LAMP products from the target and competitor. We amplified the target and competitor by LAMP under isothermal conditions with high specificity, efficiency, and rapidity and calculated the starting quantity of the target from the fluorescence intensities at the beginning and end of LAMP. We call this technique alternately binding quenching probe competitive LAMP (ABC-LAMP). We quantified amoA, which encodes the ammonia-oxidizing enzyme in environmental bacteria, as a model target by ABC-LAMP, real-time PCR, and real-time turbidimetry of LAMP. By comparison, the accuracy of ABC-LAMP was found to be similar to that of real-time PCR. Moreover, ABC-LAMP enables the accurate quantification of DNA in the presence of DNA amplification inhibitors such as humic acid, urea, and Triton X-100 that compromise the values measured by real-time PCR and real-time turbidimetry of LAMP.     

Droplet and Microchamber‐Based Digital Loop‐Mediated Isothermal Amplification (dLAMP)

Digital loop‐mediated isothermal amplification (dLAMP) refers to compartmentalizing nucleic acids and LAMP reagents into a large number of individual partitions, such as microchambers and droplets. This compartmentalization enables dLAMP to be an excellent platform to quantify the absolute number of the target nucleic acids. Owing to its low requirement for instrumentation complexity, high specificity, and strong tolerance to inhibitors in the nucleic acid samples, dLAMP has been recognized as a simple and accurate technique to quantify pathogenic nucleic acid. Herein, the general process of dLAMP techniques is summarized, the current dLAMP techniques are categorized, and a comprehensive discussion on different types of dLAMP techniques is presented. Also, the challenges of the current dLAMP are illustrated together with the possible strategies to address these challenges. In the end, the future directions of the dLAMP developments, including multitarget detection, multisample detection, and processing nucleic acid extraction are outlined. With recently significant advances in dLAMP, this technology has the potential to see more widespread use beyond the laboratory in the future.     

Integrated glass microdevice for nucleic acid purification, loop-mediated isothermal amplification, and online detection

A microdevice made of glass for genetic analysis has been fabricated, for the first time, for integration of extraction of nucleic acids and loop-mediated isothermal amplification (LAMP), followed by online fluorescence detection of amplification products on a single chip. The nucleic acid (NA) extraction region consists of a microfabricated serpentine channel in which micropillars were etched to increase the channel surface area and the capture efficiency of NAs. Nucleic acid molecules were bound to these pillars and channel surface in the presence of the chaotropic salt guanidine hydrochloride and eluted into a downstream amplification chamber with low ionic strength buffer where loop-mediated isothermal amplification was efficiently performed. Amplification can be detected online by the increase of fluorescence intensity at 540 nm when a low concentration of SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. Flow control was accomplished by using laminar flow and differential channel flow resistances. Through passivation of the LAMP chamber and the channel between the extraction region and amplification domain, effective nucleic acid extraction and amplification were performed by just using a double-channel syringe pump and a heating block. By using this integrated microdevice, the purification of nucleic acids from complex biological matrixes and their subsequent amplification and detection online could be finished within 2 h.     

Microfluidic devices for bioapplications

Harnessing the ability to precisely and reproducibly actuate fluids and manipulate bioparticles such as DNA, cells, and molecules at the microscale, microfluidics is a powerful tool that is currently revolutionizing chemical and biological analysis by replicating laboratory bench-top technology on a miniature chip-scale device, thus allowing assays to be carried out at a fraction of the time and cost while affording portability and field-use capability. Emerging from a decade of research and development in microfluidic technology are a wide range of promising laboratory and consumer biotechnological applications from microscale genetic and proteomic analysis kits, cell culture and manipulation platforms, biosensors, and pathogen detection systems to point-of-care diagnostic devices, high-throughput combinatorial drug screening platforms, schemes for targeted drug delivery and advanced therapeutics, and novel biomaterials synthesis for tissue engineering. The developments associated with these technological advances along with their respective applications to date are reviewed from a broad perspective and possible future directions that could arise from the current state of the art are discussed.     

Magnetic Graphitic Nanocapsules for Programmed DNA Fishing and Detection

Graphene nanomaterials are typically used in biosensing applications, and they have been demonstrated as good fluorescence quenchers. While many conventional amplification platforms are available, developing new nanomaterials and establishing simple, enzyme‐free and low‐cost strategies for high sensitivity biosensing is still challenging. Therefore, in this work, a core–shell magnetic graphitic nanocapsule (MGN) material is synthesized and its capabilities for the detection of biomolecules are investigated. MGN combines the unique properties of graphene and magnetic particles into one simple and sensitive biosensing platform, which quenches around 98% of the dye fluorescence within minutes. Based on a programmed multipurpose DNA capturing and releasing strategy, the MGN sensing platform demonstrates an outstanding capacity to fish, enrich, and detect DNA. Target DNA molecules as low as 50 pM could be detected, which is 3‐fold lower than the limit of detection commonly achieved by carbon nanotube and graphene‐based fluorescent biosensors. Moreover, the MGN platform exhibits good sensing specificity against DNA mismatch tests. Overall, therefore, these magnetic graphitic nanocapsules demonstrate a promising tool for molecular disease diagnosis and biomedicine. This simple fishing and enrichment strategy may also be extended to other biological and environmental applications and systems.     

Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip

An electrochemical DNA chip using an electrochemically active intercalator and DNA probe immobilized on a gold electrode has been developed for genetic analysis. In this study, the six polymorphisms associated with rheumatoid arthritis (RA), N-acetyltransferase2 (NAT2) gene polymorphisms T341C, G590A, and G857A, methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms C677T and A1298C, and serum amyloid A1 (SAA1) gene promoter polymorphism C-13T were simultaneously detected by the electrochemical DNA chip and the loop-mediated isothermal amplification (LAMP) method, which is a novel technique for DNA amplification. Human genomic DNAs were extracted from blood, and the targets containing the six polymorphisms were amplified by the LAMP method. A sample containing the six LAMP products was reacted with the electrochemical DNA chip using a DNA detection system that controls hybridization reaction, washing, electrochemical detection, and data analysis automatically. A total of 31 samples were genotyped by this method, and the results were completely consistent with those determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis or the PCR direct sequence analysis. The time required for this method was only 2 h, and operations were very simple. Therefore, this method is expected to contribute to personalized medicine based on genotype.     

LAMP实时浊度法快速检测大肠杆菌方法的建立与优化研究

根据大肠杆菌的特异性基因（malB）,设计与筛查环介导等温扩增（LAMP）的引物,通过对甜菜碱用量、环引物的浓度、反应温度、dNTPs用量等反应条件的优化,得到较佳的LAMP反应体系,建立了LAMP实时浊度法快速检测大肠杆菌;在较优条件下,先后对9株非大肠杆菌菌株进行特异性检测和5株大肠杆菌的进行同源性检测。此外,结合课题组自行研制的多通道浊度仪,对LAMP扩增产物进行实时浊度监测。结果表明：LAMP实时浊度法检测大肠杆菌的灵敏度达16.010 ng/L,与实时荧光定量PCR的检测限相当。因此,建立与优化LAMP实时浊度法可为食品中大肠杆菌的现场快速检测提供了有力手段。     

Influences of gold and silver nanoparticles in loop-mediated isothermal amplification reactions

Loop-mediated isothermal amplification (LAMP) performed with protein DNA polymerase Bst and DNA chains was influenced by nanoparticles in different ways. The effects of different concentrations of gold nanoparticles (AuNPs) with diameters 10 and 20?nm and silver nanoparticles (AgNPs) 1–10?nm in diameter on the amplification of the pR72H gene of Vibrio parahaemolyticus were investigated. AuNPs with a diameter of 10?nm in 0.6–60?nM concentration accelerated initiation of the LAMP reaction, 3?nM AuNPs reduced the reaction time by about 10?min, whereas 20?nm AuNPs did not, although neither size increased the yield after 60?min. AgNPs inhibited the LAMP reaction both in speed and yield at concentrations of 0.6–60?nM; the yield of amplification was reduced by 50% and 80% for 12 and 60?nM, respectively, after reaction for 1?h. This indicated that strong bactericidal effects of silver are also observed in its nanoparticles. The molecular mechanism of AuNPs and AgNPs in LAMP needs to be explored further, although their size-related electronic, magnetic and optical properties, as well as their ability to affect protein denaturation, or hydrophilic/hydrophobic effects may be involved.     

基于节段模型试验的千米级摩天大楼行人风环境特性研究

某千米级摩天大楼是由正三角形布置的外围3个泪滴型塔楼和中心1个圆形塔楼组成的巨型组合结构,沿其高度每隔100 m共设置10个室外平台将此4个塔楼连接起来,用于安全疏散时行人通行。由于两相邻平台之间无任何遮挡,且高空来流风速较大,给行人的舒适性与安全性带来极大的隐患,因此有必要对室外平台的行人风环境特性进行研究。考虑到风洞阻塞率的限制和室外平台上测点数量的布置要求,该文设计制作了一缩尺比为1/300的三平台式节段模型,并进行风环境试验,研究了室外平台行人高度的平均风速与阵风风速特性,并探讨不同高度和形式的挡风板对风环境特性的改善效果,为该摩天大楼的行人风环境评估奠定基础。结果表明:外围塔楼与中心塔楼之间存在明显的"狭管效应",导致行人高度风速比增大,基准模型的最大平均风速比和最大阵风风速比分别达到1.49和1.72。不同挡风板措施,不论在降低行人高度最大风速比,还是改善行人风环境的整体质量,均有效果。其中,挡风板高度或挡风板+导（抑）流板的总高度为20 mm时效果更为显著。     

气体光学检测技术及其应用研究进展

气体的快速识别与检测已成为国内外研究者迫切解决的重大问题。随着光学技术的快速发展,气体光学检测技术以其高效率、多组分、高灵敏度等显著优势而成为气体检测领域的重要研究热点之一。本文介绍了气体光学检测技术的理论基础,并按主动式与被动式两大类综述了各种典型气体光学检测技术的工作原理及应用进展。运用这些气体检测技术,已经对几十种气体实现远距离、高灵敏度的连续实时监测,完成了多种场景下对气体成分、浓度、温度等参数的测量,有效减少了危险事故的发生。通过总结和分析现有气体光学检测技术仍存在的技术问题,对未来的发展趋势进行了展望。     

Discrimination of bacteria and bacteriophages by Raman spectroscopy and surface-enhanced Raman spectroscopy

Detection of pathogenic organisms in the environment presents several challenges due to the high cost and long times typically required for identification and quantification. Polymerase chain reaction (PCR) based methods are often hindered by the presence of polymerase inhibiting compounds and so direct methods of quantification that do not require enrichment or amplification are being sought. This work presents an analysis of pathogen detection using Raman spectroscopy to identify and quantify microorganisms without drying. Confocal Raman measurements of the bacterium Escherichia coli and of two bacteriophages, MS2 and PRD1, were analyzed for characteristic peaks and to estimate detection limits using traditional Raman and surface-enhanced Raman spectroscopy (SERS). MS2, PRD1, and E. coli produced differentiable Raman spectra with approximate detection limits for PRD1 and E. coli of 10(9) pfu/mL and 10(6) cells/mL, respectively. These high detection concentration limits are partly due to the small sampling volume of the confocal system but translate to quantification of as little as 100 bacteriophages to generate a reliable spectral signal. SERS increased signal intensity 10(3) fold and presented peaks that were visible using 2-second acquisitions; however, peak locations and intensities were variable, as typical with SERS. These results demonstrate that Raman spectroscopy and SERS have potential as a pathogen monitoring platform.     

High-throughput microfluidic systems for disease detection

Accuracy and rapid response are critical to the detection of an acute infectious disease, not only because the detection results can affect the medical treatment, but also can prevent disease outbreaks. Since the current culture-based technology is time consuming and experience dependent, academia and industrial researchers are using microfluidics and nucleic acids as the fundamental ideas to build pioneering tools against infectious disease. While many point-of-care microfluidic systems have been realized to execute nucleic acid applications, high-throughput microfluidic systems are under development for various nucleic acid applications because of high efficiency and demand from the market. Building a high-throughput system is an interdisciplinary challenge because of the design concerns from science and the manufacturing concerns from engineering, but its realization will be a milestone. This article is aimed to review three essential steps of the nucleic acid-based detection realized in high-throughput formats, including polymerase chain reaction, capillary electrophoresis, and nucleic acid purification.     

Immunoassays with protein misfolding cycle amplification: a platform for ultrasensitive detection of antigen

Protein misfolding cycle amplification (PMCA), a novel technology on amplifying cyclically misfolded proteins in vitro, is conceptually analogous to DNA amplification by polymerase chain reaction (PCR) and has tremendous implications for the researches and diagnosis. Here we first introduce the protein amplification technology into the classic immunoassay and develop a PMCA-based immunoassay (immuno-PMCA) for highly sensitive detection of antigen. This method takes advantage of sandwich binding of two affinity aptamers for increased specificity, magnetic nanoparticles for fast magnetic separation, PMCA for signal amplification, and conjugated polyelectrolytes for visual detection, allowing the detection limit of antigen by colorimetry down to femtomolar level with a wide linear range from 10 to 10(4) fM. More importantly, no specialized facilities or enzymes are needed either in the amplification reaction or the evaluation of results, which indicates its great potential application in immunological research and clinical diagnostics.     

Nanomaterial-based amplified transduction of biomolecular interactions

This article reviews progress in the development of nanomaterials for amplified biosensing and discusses different nanomaterial-based bioamplification strategies. Signal amplification has attracted considerable attention for ultrasensitive detection of disease markers and biothreat agents. The emergence of nanotechnology is opening new horizons for highly sensitive bioaffinity and biocatalytic assays and for novel biosensor protocols that employ electronic, optical, or microgravimetric signal transduction. Nucleic acids and antibodies functionalized with metal or semiconductor nanoparticles have been employed as amplifying tags for the detection of DNA and proteins. The coupling of different nanomaterial-based amplification platforms and amplification processes dramatically enhances the intensity of the analytical signal and leads to ultrasensitive bioassays. The successful realization of the new nanoparticle-based signal amplification strategies requires proper attention to nonspecific adsorption issues. The implications of such nanoscale materials on amplified biodetection protocols and on the development of modern biosensors are discussed.     

Surface plasmon resonance biosensor for rapid label-free detection of microribonucleic acid at subfemtomole level

Microribonucleic acids (miRNAs) have been linked with various regulatory functions and disorders, such as cancers and heart diseases. They, therefore, present an important target for detection technologies for future medical diagnostics. We report here a novel method for rapid and sensitive miRNA detection and quantitation using surface plasmon resonance (SPR) sensor technology and a DNA*RNA antibody-based assay. The approach takes advantage of a novel high-performance portable SPR sensor instrument for spectroscopy of surface plasmons based on a special diffraction grating called a surface plasmon coupler and disperser (SPRCD). The surface of the grating is functionalized with thiolated DNA oligonucleotides which specifically capture miRNA from a liquid sample without amplification. Subsequently, an antibody that recognizes DNA*RNA hybrids is introduced to bind to the DNA*RNA complex and enhance sensor response to the captured miRNA. This approach allows detection of miRNA in less than 30 min at concentrations down to 2 pM with an absolute amount at high attomoles. The methodology is evaluated for analysis of miRNA from mouse liver tissues and is found to yield results which agree well with those provided by the quantitative polymerase chain reaction (qPCR).     

Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip

Multiplex gene assay is a valuable molecular tool not only in academic science but also in clinical diagnostics. Multiplex PCR assays, DNA microarrays, and various nanotechnology-based methods are examples of major techniques developed for analyzing multiple genes; none of these, however, are suitable for point-of-care diagnostics, especially in resource-limited settings. In this report, we describe an octopus-like multiplex microfluidic loop-mediated isothermal amplification (mμLAMP) assay for the rapid analysis of multiple genes in the point-of-care format and provide a robust approach for predicting viruses. This assay with the ability of analyzing multiple genes qualitatively and quantitatively is highly specific, operationally simple, and cost/time-effective with the detection limit of less than 10 copies/μL in 2 μL quantities of sample within 0.5 h. We successfully developed a mμLAMP chip for differentiating three human influenza A substrains and identifying eight important swine viruses.     

纳米孔传感技术应用于肿瘤早期诊断的研究进展

近几十年来,尽管研究人员一直在努力开发新的诊断和治疗技术,癌症仍然是世界上发病率和死亡率最高的疾病之一。其中关键点是在肿瘤发生的早期进行诊断。现有的肿瘤早期诊断方法有很多缺点,比如对病患肿瘤组织的侵入。因此,与肿瘤相关的无创诊断的研究已经越来越多并已经取得进展。研究发现,蛋白、DNA或者RNA等生物大分子都有可能成为潜在的肿瘤标志物,已有的检测方法也存在许多缺点。纳米孔因具有独特的物理和电学性质,对生物分子的检测有快速、无需标记和扩增等优点,已被广泛使用和证明。在不远的将来,应用纳米孔对肿瘤标志物进行检测,进行肿瘤诊断并监控治疗过程值得期待。主要介绍了纳米孔传感技术应用于肿瘤早期诊断中肿瘤标志物检测的研究进展。     

Simple bead assay for detection of live bacteria (Escherichia coli)

Bead assays are an important rapid microbial detection technology suitable for extremely low pathogen levels. We report a bead assay for rRNA extracted from Escherichia coli K12 that does not require amplification steps and has readout on an Agilent 2100 Bioanalyzer flow cytometry system. Our assay was able to detect 125 ng of RNA, which is 16 times less than reported earlier. The specificity was extremely high, with no binding to a negative control organism (Bacillus subtilis). We discuss challenges faced during optimization of the key assay components, such as varying amounts of RNA in the samples, number of beads, aggregation, and reproducibility.