Research progress and prospects of nucleic acid isothermal amplification technology

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

Nickase-dependent isothermal DNA amplification

We developed a nicking endonuclease dependent DNA amplification (NDA), using Nt.BstNBI to catalyze single-stranded nick on double-stranded DNA, and Bst DNA polymerase to make extension while sealing the nick and displacing the downstream strand. The displaced single-stranded DNA thereby serves as template for primers hybridization and extension, resulting in exponential synthesis of target DNA under isothermal condition. Over 105 folds target DNA amplification can be achieved in 30 minutes, generating DNA product suitable for both diagnosis and DNA cloning. This NDA strategy does not require thermal cycling or prerequisite nucleotides modification, making it suitable for application in the field and at the point-of-care.

Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay

When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.

Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica

Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

Sensitive and rapid detection of two toxic microalgae Alexandrium by loop-mediated isothermal amplification

A loop-mediated isothermal amplification （LAMP） assay was designed and evaluated for rapid de- tection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning （PSP）. Two sets of four specific primers targeting these two species were derived from the sequence of internal transcribed spacer （ITS） of ribosomal DNA. The method worked well in less than an hour under isothermal conditions of 65℃. LAMP specificity was validated in closely related algae as a comparison, suggesting the strict specificity of the LAMP primers. Two visual inspection approaches were feasible to interpret the positive or negative results. The detection lim- its of A. catenella and A. minutum samples using the LAMP assay were found to be 5.6 and 4.5 pg DNA, respectively. The sensitivity of this LAMP assay was 10 or 100-fold higher than Polymerase Chain Reaction （PCR） method in detecting the two microalgae. These characteristics of species specificity, sensitivity, and rapidity suggest that this method has the potentiality in the monitoring of red tide caused by A. catenella and A. minutum.

DETECTION AND APPLICATION OF MICROFLUIDIC ISOTHERMAL AMPLIFICATION ON CHIP

Loop-mediated isothermal amplification(LAMP)is a novel nucleic acid amplification method.Compared with the widely utilized polymerase chain reaction(PCR),LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different temperatures.In this paper,we designed and fabricated different kinds of polycarbonate(PC)microfluid chips,explored appropriate reaction condition for LAMP in microenvironment(1 nL→10μL),and developed a microfluidic isothermal amplification detection system.The DNA optimal amplification temperature is obtained;the starting time of exponential amplification of DNA is put forward farther.The optimal condition of DNA amplification in microenvironment,with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.

FTA-环介导等温扩增技术直接提取变异链球菌DNA的效果评价

背景:变异链球菌是龋病的重要病原菌,及时检测变异链球菌水平对龋病的早发现、早治疗有重要意义。目的:建立并评价FTA-环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术直接提取变异链球菌DNA的应用效果。方法:①制备含有ATCC标准菌株变异链球菌的菌悬液,接种于脑心浸出液培养基,充分混匀后按10倍梯度稀释成7种浓度(4.2×10^(7),4.2×10^(6),4.2×10^(5),4.2×10^(4),4.2×10^(3),4.2×10^(2),4.2×10 CFU/mL),每个稀释级做2个平行对照,并增加无菌水作为空白对照;②分别采用FTA卡、常规煮沸法、试剂盒提取及裂解液提取4种方法直接提取菌株DNA,通过LAMP技术进行扩增,并进行特异性试验,比较4种提取方法的差异。结果与结论:①4种方法提取的DNA均满足LAMP扩增的要求;②特异性试验结果显示,只有变异链球菌才可特异扩增出靶基因;③裂解液提取法最低检测限为4.2×10^(3) CFU/mL,FTA卡提取法最低检测限为4.2×10^(4) CFU/mL,试剂盒提取法和常规煮沸法最低检测限分别为4.2×10^(6) CFU/mL和4.2×10^(7) CFU/mL;④4种提取方法其他方面的比较显示,试剂盒提取法的实验成本、步骤数和时间都是最高;其他3种方法步骤数一致,其中FTA卡所需仪器设备最少,常规煮沸法单次成本最低,裂解液提取法所需时间最少;FTA卡和裂解液提取法仅需少量菌即可提取成功,后者在时间方面优于FTA卡,但相较于FTA卡其单次成本高,所需设备多;⑤结果说明,该研究建立的FTA-LAMP技术具有操作简便、特异性强、灵敏度高、结果可视化等优势,有望为高效提取检测变异链球菌提供新途径。

A photo-elutable and template-free isothermal amplification strategy for sensitive fluorescence detection of 5-formylcytosine in genomic DNA

5-Formylcytosine(5fC), as an important epigenetic modification, plays a vital role in diverse biological processes and multiple diseases by regulating gene expression. Owing to the extremely low abundance of 5fC in all mammalian tissues and high structural similarity with other cytosine derivatives, the precise and sensitive detection of 5fC is challenging. Herein, a photo-elutable and template-free isothermal amplification strategy has been proposed for the sensitive detection of 5fC in genomic DNA based on5fC-specific biotinylation, enrichment, photocleavage, and terminal deoxynucleotidyl transferase(Td T)-assisted fluorescence signal amplification, which is termed 5fC-PTIAS. By introducing the highly specific chemolabeling and the one-step photoelution processes, this strategy possesses a minimal nonspecific background as well as a much higher amplification efficiency. With the high signal-to-noise ratio, this strategy can achieve the accurate quantification of 5fC in various biological samples including mouse brain, kidney, and liver, with a limit of detection(LOD) of 0.025‰ in DNA(S/N=3). These results not only confirm the widespread distribution of 5fC but also indicate its significant variation in different tissues and ages. The bisulfite-and mass spectrometry-free strategy is highly sensitive, selective, and easily mastered, holding great promise in detecting other epigenetic modifications with much lower levels.

环介导同温DNA扩增技术鉴定血吸虫感染性钉螺方法的建立

目的建立一种快速鉴定血吸虫感染性钉螺的环介导同温DNA扩增方法。方法选择一个高拷贝的日本血吸虫基因SjR2的部分DNA序列作为扩增靶位,根据环介导同温DNA扩增原理设计合成引物,建立扩增该片段的环介导同温DNA扩增方法。使用此方法与聚合酶链反应(PCR)方法同时检测血吸虫感染性钉螺与正常钉螺的DNA,观察其应用价值。结果日本血吸虫SjR2基因的839～1 138 bp区段被成功扩增和克隆。利用根据该片段DNA序列设计合成的环介导同温DNA扩增反应的引物,成功地建立了能扩增该片段的环介导同温DNA扩增方法,其敏感性高于PCR法,能检出1 pg的血吸虫DNA。用此法检测30只感染性钉螺,阳性率为93.33%,而PCR的阳性率83.33%,两者差异有统计学意义(P<0.01)。结论环介导同温DNA扩增检测血吸虫感染性钉螺具有较高的敏感性,是一种潜在有效的血吸虫感染性钉螺鉴定方法。

单核细胞增生李斯特氏菌依赖解旋酶DNA恒温扩增检测方法的建立

为建立单核细胞增生李斯特氏菌(Lm)的依赖解旋酶DNA恒温扩增(HDA)检测方法,本研究以Lm的iap基因为目的片段设计特异性引物,建立了可在65℃恒温扩增快速(90 min)检测Lm的HDA检测方法,分别对反应体系中的MgAc2、Bst聚合酶、UvrD解旋酶、dNTPs以及引物等的浓度进行优化,进行了特异性和灵敏度试验,并与普通PCR方法进行了比较。结果表明,所建立起的检测法最低检测限为7.8×103 cfu/mL,灵敏度与普通PCR方法相当。Lm的HDA检测方法具有普通PCR的特异、灵敏等特点,并且对仪器要求更低,具有良好的应用前景。

应用依赖解旋酶DNA恒温扩增技术检测副溶血性弧菌

以副溶血性弧菌的tlh基因为目的片段设计特异性引物,成功建立了恒温条件下快速检测副溶血性弧菌的依赖解旋酶DNA恒温扩增技术(HDA)的检测法,进行了特异性和灵敏度实验,并与普通PCR方法进行了比较。该HDA检测方法最低检测限为19.9ng·mL-1,与普通PCR方法相当。副溶血性弧菌的HDA检测方法具有普通PCR的特异、灵敏等特点,并且对仪器要求更低,用普通水浴槽即可进行反应。

利用模拟酶催化显色和等温核酸扩增反应检测特定序列DNA

基于血红素（Hemin）与G-四联体（G4）所形成模拟酶的催化作用,结合等温指数扩增反应（IEX-PAR）,建立了特定序列DNA的显色检测方法.目标DNA引发IEXPAR,通过设计模板序列,IEXPAR产生大量富含鸟嘌呤的单链DNA,在K＋存在时,该单链DNA可形成G4结构,G4可以与Hemin结合形成具有过氧化物酶活性的模拟酶（Hemin-G4）,Hemin-G4催化H2 O2氧化2,2-联氮-二（3-乙基-苯并噻唑-6-磺酸）二铵盐（ABTS）,使体系颜色发生变化,反应产物最大吸收波长为421 nm.对显色反应体系中的实验参数进行了优化,包括Hemin浓度、ABTS浓度、H2 O2浓度、IEXPAR时间和显色反应时间.在最佳条件下,所建立的体系可以简便、快速检测0.1 ～ 10 nmol/L的目标DNA分子,且本方法能够很好地区分单个碱基的差别,具有良好的特异性.

环介导等温扩增(LAMP)试剂盒检测石蜡包埋肝组织中乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)

目的建立一种检测石蜡包埋的肝组织中乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)的新方法,并探讨其应用价值和临床意义。方法提取30例慢性HBV感染患者的石蜡包埋肝组织中的DNA,采用质粒安全ATP依赖性DNA酶(plasmid-safe ATP-dependent DNase,PSAD)消化,去除非cccDNA后,加入引物及Bst DNA酶进行环介导等温扩增(loop-mediated isothermal amplification,LAMP),将检测结果与临床实验室资料结合并进行分析,以判断其临床应用价值。结果 30例HBV感染患者中,有17例HBV cccDNA阳性,阳性检测率56.67%。其中肝癌5例、肝硬化4例、重型乙型肝炎5例、慢性乙型肝炎(轻-中度)3例。提示肝组织HBV cccDNA阳性可能与疾病进展密切相关,与血清HBV cccDNA阳性有一定的相关性,与乙型肝炎病毒血清免疫标志物以及肝功能状况无明显相关。结论 LAMP法能检测到石蜡包埋肝组织中的HBV cccDNA,结合临床实验室资料,对判断抗病毒药物疗效及研究乙型肝炎致病机制具有一定的意义。

脱氧核酶催化放大效应在微RNA传感中的研究与应用

微RNA(microRNAs,miRNAs)是一种与肿瘤等多种疾病的发生发展密切相关的短序列非编码RNA,其具有丰度低、序列同源性高和易降解等特点,因此,miRNAs的高灵敏、高特异检测面临巨大挑战。具有RNA切割活性的脱氧核酶是新兴的一类功能性单链DNA分子,此类酶能在金属离子的辅助下对核酸底物特异性切割,释放miRNA进行循环再利用,实现信号放大。目前,基于脱氧核酶催化放大检测miRNA的新方法研究是近年来科研人员的重点关注方向之一。本文根据脱氧核酶结合不同的生物传感新技术和新材料,对近年来发展出的基于脱氧核酶催化放大检测miRNAs的新方法进行分类与回顾,归纳为脱氧核酶DNA自组装、脱氧核酶偶联等温扩增以及脱氧核酶结合新型纳米材料的3类方向,并逐一阐述各个方向的基本原理及其在生物传感领域、医学检测方向的最新研究进展与应用实例,旨在为进一步的精准灵敏检测miRNAs新策略研究提供参考。

银杏叶片DNA提取及PCR和LAMP鉴定技术

以中药材银杏叶为试材,采用环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法进行银杏叶真伪鉴定。设计LAMP反应引物,并分别对银杏叶及其混伪品进行LAMP引物特异性试验和LAMP引物灵敏度试验。结果表明:GB1能够有效区分银杏叶样品与常见混伪品,最低检出浓度为4.6×10^(-5 )ng·μL^(-1),灵敏度是普通PCR的10倍。从而建立了快速有效鉴定银杏叶的LAMP方法。

DNA检测技术在鳕鱼及其制品鉴定中的应用

鳕鱼是经济价值高和营养价值高的重要食用鱼类,但由于市场上鱼类俗名混乱问题,以及不同鱼种之间价值的巨大差异,错误标识鳕鱼产品或以次充好的现象时有发生,有可能因为含有过敏原成分或有毒成分造成食品安全风险。因此,需要快速可靠的鳕鱼成分鉴定方法和来保障市场监管。目前,鳕鱼及其制品的鉴定方法主要为DNA检测技术。本文主要介绍了DNA指纹图谱限制性片段长度多态性聚合酶链反应(cleaved amplification polymorphism sequence-tagged sites, PCR-RFLP)技术、DNA条形码技术、环介导等温扩增技术和实时荧光定量PCR技术的原理、优缺点、在鳕鱼及其制品鉴定中的应用,最后讨论了鳕鱼及其制品鉴定方法的发展趋势和监管建议。

DNA信息存储中关键生化方法的研究

随着生物技术特别是高通量的DNA合成和测序技术的发展,DNA信息存储技术在存储容量、稳定性以及可重复读取等方面都取得了重大成就。但目前携带有数据信息的大规模寡核苷酸文库的生化技术的操纵仍面临着巨大的挑战,比如合成、扩增、保存或测序过程引起的寡核苷酸不均一性、DNA序列丢失、碱基突变、DNA分子的衰减等,这些因素限制了DNA信息存储走向实际工业化应用。本文从操纵大型寡核苷酸文库的生化技术角度出发,归纳总结了造成生化问题的原因以及为解决这些问题所开发的一系列方法,包括合成工艺的改进、寡核苷酸文库的均一化、多种DNA保存方法、变体PCR以及恒温扩增反应,论证了它们在DNA信息存储流程中避免上述生化问题的可行性与有效性,最后分析了现阶段DNA信息存储所面临的合成、长时间保存方法以及扩增等方面的挑战。本文旨在为实现对大型寡核苷酸文库的操纵奠定基础,以期促进DNA信息存储迈向实际应用。

发酵乳中植物乳杆菌依赖解旋酶恒温基因扩增检测方法的建立

通过依赖解旋酶恒温基因扩增(helicase-dependent isothermal DNA amplification,HDA)技术建立一种快速检测发酵乳中植物乳杆菌的方法。根据植物乳杆菌scrB基因序列(基因号为AJ579541.1)设计特异性引物,通过实验筛选出检测体系中UvrD解旋酶和T4 gp32蛋白的较优含量,达到最优反应条件。所建立的HDA方法通过人工添加植物乳杆菌确定检出限,通过多种菌株扩增验证方法特异性,通过扩增产物电泳和序列比对分析验证方法一致性和稳定性。结果表明:采用HDA法检测发酵乳中植物乳杆菌,反应体系中UvrD解旋酶和T4 gp32蛋白的适宜添加量分别为0.15μg和5.0μg,特异性良好,与涉及的其他菌株均未发生扩增,检出限为2.8×10^(1) CFU/g,扩增序列与设计序列长度(273 bp)一致且序列同源性为100%;本研究建立的方法可以快速检测发酵乳中植物乳杆菌,具有灵敏度高、操作简单、应用前景广等特点。

基于双富集微流控技术的食品中蜡样芽孢杆菌快速检测

为实现食源性致病菌多场景即时检验,替换常规检验及快检技术中耗时的增菌培养过程,以缩短检验时间和降低实验环境及设备要求。研究以蜡样芽孢杆菌为研究对象,采用双层膜富集样品中菌体,磁珠提取纯化微量DNA的方式,取代增菌培养过程,同时将环介导等温扩增反应与微流控芯片相结合,有效避免扩增中气溶胶污染问题。实验结果显示:使用双富集微流控技术检测食品中蜡样芽孢杆菌,可将现行国标的检验时间由5~7 d缩短至1 h内,方法特异性良好,检出限为10 CFU/g(mL)等同于现行国标,Ct值变异系数<5%,显示重复性好。研究建立的双富集微流控技术快速检测食品中蜡样芽孢杆菌方法可实现食源性致病菌即时快速检验,为食源性致病菌引起的食品安全事件和舆情风险防控快速反应提供技术支撑。

新型恒温核酸扩增法快速检测副溶血性弧菌的研究

利用DNA环介导恒温核酸扩增法(loop-mediated isothermal amplification of DNA,LAMP)设计一对外引物和一对内引物,通过引物特异性识别tlh基因上的六个独立区域来快速检测副溶血弧菌。同时将检测结果与PCR方法进行比较。结果表明,LAMP反应在65℃恒温条件60min内完成,凝胶电泳呈现梯型条带;肉眼观察阳性结果出现白色混浊现象,添加1×SYBRGreenI荧光染料后,绿色的阳性结果很明显区别于橙色阴性结果;LAMP方法的最低检出限为10CFU/ml,PCR方法为103CFU/ml,LAMP方法检测灵敏度是PCR方法的100倍。因此,LAMP方法用于快速检测副溶血弧菌具有检测过程简单、反应结果肉眼即辨别并且灵敏度高特异性强的特点。实验装置简便,能够提供稳定热源即可,所以LAMP方法特别适合于现场快速诊断。