DNA circuits as amplifiers for the detection of nucleic acids on a paperfluidic platform

This article describes the use of non-enzymatic nucleic acid circuits based on strand exchange reactions to detect target sequences on a paperfluidic platform. The DNA circuits that were implemented include a non-enzymatic amplifier and transduction to a fluorescent reporter; these yield an order of magnitude improvement in detection of an input nucleic acid signal. To further improve signal amplification and detection, we integrated the enzyme-free amplifier with loop-mediated isothermal amplification (LAMP). By bridging the gap between the low concentrations of LAMP amplicons and the limits of fluorescence detection, the non-enzymatic amplifier allowed us to detect as few as 1200 input templates in a paperfluidic format.     

Pullulan reduces the non-specific amplification of loop-mediated isothermal amplification (LAMP)

Loop-mediated isothermal amplification (LAMP) has been developed as a versatile method for nucleic acid analysis in many applications. However, non-specific LAMP leading to false-positive outcomes has been observed frequently. To solve this problem, we selected six molecules as the additives for evaluating their effects on the improvement of the LAMP specificity. Experimental results show that bovine serum albumin (BSA) and DL-dithiothreitol (DTT) have negative effects on the LAMP specificity; dimethyl sulfoxide (DMSO), tetramethylene sulfoxide (TMSO), and glycerol could inhibit the non-specific LAMP moderately. Surprisingly, pullulan shows an ability to inhibit the non-specific amplification of LAMP significantly without affecting the sample amplification of LAMP, and this inhibitory effect is concentration dependent. Thus, pullulan could be considered as the most promising additive to improve the amplification specificity in the LAMP-based detection and analysis of nucleic acids.

     

Mathematical model to reduce loop mediated isothermal amplification (LAMP) false‐positive diagnosis

Loop mediated isothermal amplification (LAMP) is a nucleic acid amplification technique performed under isothermal conditions. The output of this amplification technique includes multiple different sizes of deoxyribonucleic acid (DNA) structures which are identified by a banding pattern on gel electrophoresis plots. Although this is a specific amplification technique, the complexity of the primer design and amplification still lead to the issue of obtaining false‐positive results, especially when a positive reading is determined solely by whether there is any banding pattern in the gel electrophoresis plot. Here, we first performed extensive LAMP experiments and evaluated the DNA structures using microchip electrophoresis. We then developed a mathematical model derived from the various components that make up an entire LAMP structure to predict the full LAMP structure size in base pairs. This model can be implemented by users to make predictions for specific, DNA size dependent, banding patterns on their gel electrophoresis plots. Each prediction is specific to the target sequence and primers used and therefore reduces incorrect diagnosis errors through identifying true‐positive and false‐positive results. This model was accurately tested with multiple primer sets in house and was also translatable to different DNA and RNA types in previously published literature. The mathematical model can ultimately be used to reduce false‐positive LAMP diagnosis errors for applications ranging from tuberculosis diagnostics to E. coli to numerous other infectious diseases.     

Recording the Reaction Process of Loop‐Mediated Isothermal Amplification (LAMP) by Monitoring the Voltammetric Response of 2′‐Deoxyguanosine 5′‐Triphosphate

We describe here a novel strategy for recording the reaction process of loop‐mediated isothermal amplification (LAMP) by monitoring the voltammetric response of 2′‐deoxyguanosine 5′‐triphosphate (dGTP). Unlike the other three kinds of reactive substrates for DNA synthesis in LAMP reaction, dGTP exhibits sensitive voltammetric response at the carbon nanotube array electrode. When the LAMP reaction occurs, the concentration of dGTP decreases accordingly, bringing forth the decrease of the anodic peak current (i_pa). In inversion, the decrease of the i_pa of dGTP was used to characterize the reaction process of LAMP. The relationships among the LAMP reaction time, the initial quantity of template DNA and the value change of the i_pa were studied. The results indicate that the protocol integrated LAMP and voltammetric techniques can be used for not only qualitative gene discrimination but also quantitative gene assay in a wide range. The malB gene extracted from common strains of Escherichia coli cells was tested as a model. The detecting results of LAMPs obtained by voltammetric method were in good agreement with those by optical‐based methods (gel electrophoresis and fluorescent dye).     

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach

In 2003 the European Commission introduced a 0.9 % threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5 %. A false-negative rate of only 5 % for 1 % GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.     

Ultrasensitive electrochemiluminescent aptasensor for ochratoxin A detection with the loop-mediated isothermal amplification

In this study, we for the first time presented an efficient, accurate, rapid, simple and ultrasensitive detection system for small molecule ochratoxin A (OTA) by using the integration of loop-mediated isothermal amplification (LAMP) technique and subsequently direct readout of LAMP amplicons with a signal-on electrochemiluminescent (ECL) system. Firstly, the dsDNA composed by OTA aptamer and its capture DNA were immobilized on the electrode. After the target recognition, the OTA aptamer bond with target OTA and subsequently left off the electrode, which effectively decreased the immobilization amount of OTA aptamer on electrode. Then, the remaining OTA aptamers on the electrode served as inner primer to initiate the LAMP reaction. Interestingly, the LAMP amplification was detected by monitoring the intercalation of DNA-binding Ru(phen)₃²⁺ ECL indictors into newly formed amplicons with a set of integrated electrodes. The ECL indictor Ru(phen)₃²⁺ binding to amplicons caused the reduction of the ECL intensity due to the slow diffusion of Ru(phen)₃²⁺–amplicons complex to the electrode surface. Therefore, the presence of more OTA was expected to lead to the release of more OTA aptamer, which meant less OTA aptamer remained on electrode for producing LAMP amplicons, resulting in less Ru(phen)₃²⁺ interlaced into the formed amplicons within a fixed Ru(phen)₃²⁺ amount with an obviously increased ECL signal input. As a result, a detection limit as low as 10 fM for OTA was achieved. The aptasensor also has good reproducibility and stability.     

One-step ultrasensitive detection of microRNAs with loop-mediated isothermal amplification (LAMP)

An ultrasensitive microRNA assay was developed with one-step loop-mediated isothermal amplification (LAMP) initiated by the target microRNA.     

An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases

A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA?) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette's flow control. The FTA membrane also serves another critical role-enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food.     

基于毛细管电泳的环介导恒温扩增技术检测方法研究

以大肠杆菌(E.coli)为对象,采用环介导恒温扩增技术(LAMP)对其扩增,在实验室自制的毛细管电泳-诱导荧光平台上建立了LAMP产物的检测新方法。引物F3,B3,FIP,BIP扩增的E.coli LAMP产物大小为240 bp。优化的毛细管电泳条件为:毛细管有效长度/总长度(10 cm/15 cm),筛分介质溶液为0.5%羟乙基纤维素(1 300 K),电场强度(100 V/cm),进样条件(100 V/cm,1.0 s)。毛细管电泳时,DNA长度在100~500 bp范围内与其迁移时间呈线性关系,相关系数为0.996。在相同毛细管电泳条件下对E.coli LAMP产物进行分析,并利用这种线性关系在电泳图中对E.coli LAMP产物与假阳性产物做区分,结果表明,毛细管电泳技术不仅可在15 min内实现LAMP产物及附加产物的快速检测,而且可快速区分LAMP阳性及假阳性实验产物。采用建立的毛细管电泳快速检测LAMP产物的方法,对AB0174 E.coli基因实施了LAMP,结果表明该方法适合DNA LAMP产物的快速检测。     

Detection of the food allergen celery via loop-mediated isothermal amplification technique

Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0 % for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.     

Detection of LAMP Products Using CNT Array Electrode

We described here a novel strategy for discriminating target gene by integrating loop‐mediated isothermal amplification (LAMP) and differential pulse voltammetry (DPV). After a successful LAMP reaction, the anodic peak current (i_pa) of the free 2′‐deoxyguanosine 5′‐triphosphate (dGTP) decreased remarkably at a carbon nanotubes array working electrode, owing to the consumption of free dGTP as one of reactive substrates. Thus, the change of current response was used to characterize the result of LAMP reaction. And hence the presence of the target gene in template DNA could be discriminated easily. The malB gene extracted from Escherichia coli cells was tested as a model. After the reaction for 30 min, the LAMP mix was scanned directly. Then the information of the target gene in 0.8 picogram template DNA was obtained accurately. The result was in good accordance with that obtained with optical‐based methods (gel electrophoresis and fluorescent dye). The new strategy has the advantages of being very simple to perform, rapid response, elimination of post‐amplification processing, avoidance of auxiliary reagents and low cost (there was almost no cost for the detection step). Therefore, it was quite promising for use in miniaturized devices and in the development of point‐of‐care applications.     

Exponential Isothermal Amplification of Nucleic Acids and Assays for Proteins,Cells, Small Molecules,and Enzyme Activities: An EXPAR Example

Isothermal exponential amplification techniques, such as strand‐displacement amplification (SDA), rolling circle amplification (RCA), loop‐mediated isothermal amplification (LAMP), nucleic acid sequence based amplification (NASBA), helicase‐dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on‐site, point‐of‐care, and in situ assay applications. These amplification techniques eliminate the need for temperature cycling, as required for the polymerase chain reaction (PCR), while achieving comparable amplification yields. We highlight here recent advances in the exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. The incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables the highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from nonspecific template interactions, must be addressed to further improve isothermal and exponential amplification techniques.     

Applications of Loop-Mediated Isothermal DNA Amplification

During the last 10 years, with the development of loop-mediated isothermal amplification (LAMP) method, it has been widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency, and outstanding specificity. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. Expensive equipment are not necessary to acquire a high level of precision, and there are fewer preparation steps compared to conventional PCR and real-time PCR assays. This paper briefly summarized the applications of LAMP method in pathogenic microorganisms, genetically modified ingredients, tumor detection, and embryo sex identification.     

A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification

This study reports a new diagnostic assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by combing nucleic acid extraction and isothermal amplification of target nucleic acids in a magnetic bead-based microfluidic system. By using specific probe-conjugated magnetic beads, the target deoxyribonucleic acid (DNA) of the MRSA can be specifically recognized and hybridized onto the surface of the magnetic beads which are then mixed with clinical sample lysates. This is followed by purifying and concentrating the target DNA from the clinical sample lysates by applying a magnetic field. Nucleic acid amplification of the target genes can then be performed by the use of a loop-mediated isothermal amplification (LAMP) process via the incorporation of a built-in micro temperature control module, followed by analyzing the optical density (OD) of the LAMP amplicons using a spectrophotometer. Significantly, experimental results show that the limit of detection (LOD) for MRSA in the clinical samples is approximately 10 fg μL(-1) by performing this diagnostic assay in the magnetic bead-based microfluidic system. In addition, the entire diagnostic protocol, from bio-sample pre-treatment to optical detection, can be automatically completed within 60 min. Consequently, this miniature diagnostic assay may become a powerful tool for the rapid purification and detection of MRSA and a potential point-of-care platform for detection of other types of infections.     

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10⁻¹ genomic equivalent ml⁻¹. An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.     

芯片毛细管电泳用于人乳头瘤病毒分析的研究进展

人乳头瘤病毒（human papillomavirus,HPV）是一种常见的球形DNA病毒,目前已报道其可以导致6种类型的癌症发生,因此HPV病毒检测方法的研究引起了人们的重视。芯片毛细管电泳（MCE）,作为一种芯片实验设备,结合各种信号放大技术为HPV分型检测提供了简单、快速、高灵敏度和易便携化的检测方法。该文综述了MCE在常规HPV分型检测中的最新研究进展,主要分为MCE技术和MCE结合核酸扩增技术两个部分。综述的第一部分介绍了MCE系统、MCE芯片结构设计和电泳分离方法。典型的MCE系统包含了高压电源、分离芯片、电解液池、进样系统、检测系统等。该文还介绍了近年来应用最广泛的4种芯片通道,包括分离直通道、T型通道、蛇形通道以及双通道,并分别对它们的优缺点进行了比较。第二部分主要介绍芯片电泳在HPV检测中的应用和发展。由于MCE技术的应用,HPV目标物的分离时间,从以前的几个小时缩短到几分钟,极大地提高了分离速度。重点介绍了各种核酸扩增技术结合MCE检测HPV的方法。对聚合酶链式反应（PCR）和MCE结合用于HPV的检测技术、环介导等温扩增（LAMP）技术的HPV检测方法、基于PCR结合限制性片段长度多态性（RFLP）技术用于HPV分型的DNA检测、基于核酸序列扩增（NASBA）技术检测HPV mRNA、巢式PCR等进行了比较分析。其次,对HPV其他检测方法进行了总结,其中包括PCR结合傅里叶变换红外光谱法（FT-IR）、纳米技术、DNA探针结合电化学方法、亚铜粒子氧化还原锌掺杂的二硫化钼量子点结合T7外切酶电化学发光法和基于CRISPR/Cas12a的环介导等温扩增法。在这些非MCE方法中,电化学传感法,如阻抗法、脉冲伏安法和流动生物传感器,由于背景信号低、时间控制能力强,是一种比较理想的方法。最后,虽然近年来MCE技术得到了发展,所开发的设备得到了应用,但目前在MCE技术、方法和应用方面仍然存在一些挑战。MCE技术在HPV分型检测应用中面临的第一个挑战是,MCE本身无法对HPV核酸进行信号放大,从而不能在HPV的高灵敏和高选择性分析中得到很好的应用。第二个挑战是,虽然有一些研究者已经成功地将PCR和MCE集成在一个芯片上,但该技术的广泛应用仍面临困难,目前仍然没有真正集成的PCR-MCE芯片用于HPV检测。第三个挑战是目前MCE技术无法实现小型化、自动化器件的制造。最后,文章就MCE在HPV分型检测中开发更自动化、更快速以及更稳定可靠的检测技术提出了一些观点和见解,希望能对感兴趣的读者提供一些启发。     

Gold Nanoparticles Based Colorimetric Detection of Target DNA After Loop-mediated Isothermal Amplification

We have developed a rapid, simple and label-free colorimetric method for the identification of target DNA. It is based on loop-mediated isothermal amplification(LAMP). Plain gold nanoparticles(AuNPs) are used to indicate the occurrence of LAMP. The amplified product is mixed with AuNPs in an optimized ratio, at which the deoxyribonucleotides(dNTPs) bind to the AuNPs via ligand-metal interactions and thus enhance AuNPs stability. If a target DNA is amplified, the dramatic reduction of the dNTPs leads to the aggregation of AuNPs and a color change from red to blue. The success of the method strongly depends on the ionic strength of the solution and the initial concentration of dNTPs. Unlike other methods for the identification of isothermal products, this method is simple and can be readily applied on site where instrumentation is inadequate or even lacking.