环介导等温扩增技术改进的研究进展

环介导等温扩增法(LAMP)是近年发展起来的新型核酸检测技术,因其检测特异性好、灵敏度高、时间短、无需热循环设备而在病原体检测中得到广泛应用。基于该技术进行的改造和改良也层出不穷,本文从该技术的方法改造、缩短反应时间和简化模板预处理、多重扩增产物分析、防止假阳性污染四个方面对近几年LAMP技术的发展进行综述,为今后以该技术平台为基础的创新和应用提供理论依据。     

基于核酸侵入反应的生物分子检测技术的研究进展

核酸侵入反应是由5＇核酸内切酶或flap内切酶催化的,能够识别切割核酸片段形成的特异性结构的一类反应。近年来发展了很多基于该反应的生物大分子检测技术,能够对DNA、RNA、miRNA及蛋白质进行高灵敏、高特异性的测定。这些技术大都无需扩增待测靶标,极大地降低了扩增产物交叉污染的风险,在临床检测中具有很大的应用前景。本文对这些检测技术的原理及应用作简要综述。     

基于核酸侵入反应的生物分子检测技术的研究进展

核酸侵入反应是由5’核酸内切酶或flap内切酶催化的,能够识别切割核酸片段形成的特异性结构的一类反应。近年来发展了很多基于该反应的生物大分子检测技术,能够对DNA、RNA、miRNA及蛋白质进行高灵敏、高特异性的测定。这些技术大都无需扩增待测靶标,极大地降低了扩增产物交叉污染的风险,在临床检测中具有很大的应用前景。本文对这些检测技术的原理及应用作简要综述。     

等温扩增技术及其结合CRISPR在微生物快速检测中的研究进展

等温扩增技术因其对仪器依赖性低、核酸扩增高效等优势,非常适合于快速检测,已在微生物快速检测领域得到了广泛应用。本文从核酸提取、等温扩增(以环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)和重组酶聚合酶扩增技术(Recombinase polymerase amplification,RPA)为例)和产物检测角度,就近年来核酸等温扩增技术的发展及其在病原微生物核酸快速检测领域的应用进行综述,并概述了核酸等温扩增技术与CRISPR(Clustered regularly interspaced short palindromic repeats)基因编辑技术相结合的最新研究成果,为这些新兴技术的研究和未来的发展提供新思路。     

不同加热方式对嗜肺军团菌环介导等温扩增检测法的影响

目的：研究不同的加热方式对嗜肺军团菌环介导等温扩增检测法的影响方法：用已知的13株嗜肺军团菌样本,采用空气浴、水浴和PCR仪同时进行环介导等温扩增,观察沉淀反应、荧光反应以及产物电泳结果。结果：水浴和PCR仪加热LAMP反应的沉淀产物较多,荧光反应较强,电泳检测结果较为明显。空气浴的3种检测结果均较弱。结论：采用水浴和PCR仪进行环介导等温扩增反应的效果较好,从仪器设备的成本及实验条件考虑,采用水浴是环介导等温扩增反应首选的加热方式。     

环介导等温扩增技术的研究进展

环介导等温扩增(loop-mediated isothermal amplification,LAMP)是一种新式核酸扩增技术,它依靠一种具有链置换活性的DNA聚合酶和2对特殊设计的引物,不需要反复的温度循环和昂贵的仪器设备,在等温条件下即可高效快速地完成扩增反应,目前已广泛应用于细菌、病毒、寄生虫等病原体的检测,及动物胚胎性别的鉴定。该文总结了LAMP技术的基本原理、相对于传统核酸检测技术的优点、产物的检测方法及其临床应用,最后指出LAMP目前存在的不足以及采取的相应措施,并对其发展前景进行了展望。     

单管可视化环介导等温扩增技术快速检测恶性疟原虫

目的:建立一种基于环介导等温核酸扩增技术(Loop-mediated Isothermal Amplification,LAMP)的恶性疟原虫高灵敏可视化闭管检测方法。方法:针对恶性疟原虫核糖体DNA的序列保守区设计LAMP引物,通过优化LAMP体系中的Mg2+、甜菜碱浓度和反应温度等因素,建立环介导等温扩增法;并结合蜡封反应管对产物进行检测,检测结果可直接通过肉眼观察SYBR Green I荧光显色进行判定。结果:本方法可检测到70个拷贝/管的恶性疟原虫核酸片段,并具有高特异性,可区分检测常见的血液病毒。该法具有如下优点:1、整个反应恒温进行,无需热循环仪;2、闭管检测,极大降低了扩增产物交叉污染的风险;3、检测速度快,整个检测过程只需30 min。结论:该法的建立为恶性疟原虫的现场快速筛检提供了一种简便、高灵敏、高特异的工具。     

不同加热方式对嗜肺军团菌环介导等温扩增检测法的影响

目的:研究不同的加热方式对嗜肺军团菌环介导等温扩增检测法的影响方法:用已知的13株嗜肺军团菌样本,采用空气浴、水浴和PCR仪同时进行环介导等温扩增,观察沉淀反应、荧光反应以及产物电泳结果。结果:水浴和PCR仪加热LAMP反应的沉淀产物较多,荧光反应较强,电泳检测结果较为明显。空气浴的3种检测结果均较弱。结论:采用水浴和PCR仪进行环介导等温扩增反应的效果较好,从仪器设备的成本及实验条件考虑,采用水浴是环介导等温扩增反应首选的加热方式。     

逆转录环介导等温核酸扩增技术(RT-LAMP)在H5N1禽流感病毒基因检测中的应用

建立一种便捷、灵敏的检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)用于H5N1亚型禽流感病毒基因检测.该技术使用特异对应于靶序列中8个基因区段的6条特异引物,在等温条件下进行核酸扩增反应.对51份实验感染动物及病毒培养标本的H5N1亚型禽流感病毒的HA、NA基因区进行了RT-LAMP检测,并以SYBR Green Ⅰ为反应指示剂进行了逆转录环介导等温核酸扩增技术,对该反应进行实时监控,经对扩增产物做内切酶验证和测序分析,证明RT-LAMP技术的特异性;同时,用10倍系列稀释的RNA样品对该检测方法的灵敏度进行了测试.结果显示:利用RT-LAMP技术成功检测到H5N1禽流感病毒的HA、NA基因区,且RT-LAMP与Real-time PCR结果呈现很好的一致性.此方法的灵敏度可达到能检测10个拷贝RNA分子水平.因此,RT-LAMP技术应用于H5N1亚型禽流感病毒的快速检测是一种可行的方法.     

Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites

A loop-mediated isothermal amplification (LAMP) technique has been used as a novel nucleic acid detection method, whereby the target DNA can be amplified with high specificity and sensitivity under an isothermal condition using a set of four specific primers. In this study, we designed two sets of the LAMP primers for rhoptry-associated protein-1 genes of Babesia bovis and B. bigemina, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers to construct a multiplex LAMP (mLAMP) method by combining these two sets totaling eight primers. The mLAMP method was distinguishable between B. bovis and B. bigemina, simultaneously, due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 10(3) and 10(5) times higher on the detection limits for B. bovis and B. bigemina, respectively, than those of the classical PCR methods. Of 40 blood samples collected from cattle living in Ghana, 12 and 27% were positively detected by the mLAMP for B. bovis and B. bigemina, respectively. Furthermore, 14 and 23% of 90 blood samples from cattle in Zambia showed mLAMP-positive reactions to B. bovis and B. bigemina, respectively. These findings indicate that this mLAMP method is a new convenient tool for simultaneous detection of the bovine Babesia parasites.     

血源性乙型肝炎病毒、丙型肝炎病毒和艾滋病病毒核酸筛查系统的建立与应用

目的：建立同时实现乙型肝炎病毒（hepatitisBvirus,HBV）、丙型肝炎病毒（hepatitisCvirus,HCV）、艾滋病病毒（humanImmunodeficiencyVirus,HIV）检测的多重核酸筛查系统。方法：以HBV、HCV、HIV的保守序列为模板设计特异性引物和探针,通过核酸自动提取系统结合一步法RT-PCR技术平台,优化相关反应体系和条件,建立多重多色实时荧光PCR检测血源性传播病毒的核酸筛查系统。将该系统用于101387例血浆样本的筛查。结果：本研究建立的核酸筛查系统特异性好,HBV灵敏度可以达到20IU／ml,HCV灵敏度可以达到100IU／ml,HIV灵敏度可以达到50IU／mL。结论：本研究建立的核酸筛查系统具有高度自动化、高灵敏度、低成本等特点,适合我国血站系统推广使用。     

应用xMAP液态芯片多重快速检测四种病原微生物的研究

目的：建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法：根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果：xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论：xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

快速检测HBV DNA的环状介导等温DNA扩增法

环状介导等温DNA扩增（LAMP）技术是一种新的核酸扩增方法,它能够高特异性、高效、快速地进行核酸的扩增。利用LAMP法检测乙型肝炎病毒（HBV）,能够在等温条件下于1h内将少量的基因拷贝数扩增至10^9,在对65份临床标本的检测中显示了较高的特异性。与现有的PCR技术相比,LAMP法更加简便快速,且在等温条件下进行,不需要复杂的仪器设备,为临床检测乙肝病毒提供了一个快速筒便的新方法。     

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

Analysis and application of NAT in Dongguan blood center more than 10 years

This report reviewed the efficacy of nucleic acid testing (NAT), derived from assaying and measuring data, using a Cobass201 system at the Dongguan blood center from 2008 to 2017. During this period, four blood screening models, each reflecting procedure improvements designed to improve residual risk (RR) assessment were assessed. A total of 716 846 blood donors were screened, detecting 1 395 positive by the mixed pool test, which were finally modified to 900 positive cases, after final detection by the separation-single test: detecting 6 HIV cases, 4 HCV cases and 890 HBV cases with a total positive rate of 1.25%. The lowest result was obtained from the twice administered enzyme-linked immunoassay (ELISA) test,used in conjunction with the single Cobas MPX v2.0 model,with rates of: 1/7 405 for HBV, 1/346 020 for HCV and 1/473 934 for HIV, respectively. NAT positive rate is not affected by different screening models. NAT is a recent detection method which can be used to good effect with ELISA, and is a worth while procedure for promoting blood transfusion safety.     

Health economics of blood transfusion safety – focus on sub-Saharan Africa

Background and objectivesHealth economics provides a standardised methodology for valid comparisons of interventions in different fields of health care. This review discusses the health economic evaluations of strategies to enhance blood product safety in sub-Saharan Africa.MethodsWe reviewed health economic methodology with special reference to cost-effectiveness analysis. We searched the literature for cost-effectiveness in blood product safety in sub-Saharan Africa.ResultHIV-antibody screening in different settings in sub-Saharan Africa showed health gains and saved costs. Except for adding HIV-p24 screening, adding other tests such as nucleic acid amplification testing (NAT) to HIV-antibody screening displayed incremental cost-effectiveness ratios greater than the WHO/World Bank specified threshold for cost-effectiveness. The addition of HIV-p24 in combination with HCV antibody/antigen screening and multiplex (HBV, HCV and HIV) NAT in pools of 24 may also be cost-effective options for Ghana.ConclusionsFrom a health economic viewpoint, HIV-antibody screening should always be implemented in sub-Saharan Africa. The addition of HIV-p24 antigen screening, in combination with HCV antibody/antigen screening and multiplex (HBV, HCV and HIV) NAT in pools of 24 may be feasible options for Ghana. Suggestions for future health economic evaluations of blood transfusion safety interventions in sub-Saharan Africa are: mis-transfusion, laboratory quality and donor management.     

Detection of Fomitiporia torreyae and Fulviformes umbrinellus by multiplex loop‐mediated isothermal amplification (mLAMP) for diagnosis of Japanese pear dwarf

Japanese pear dwarf, caused by the fungi Fomitiporia torreyae or Fulviformes umbrinellus, is one of the most important diseases affecting Japanese pear (Pyrus pyrifolia var. culta). To diagnose this disease, a multiplex loop‐mediated isothermal amplification (mLAMP) reaction using primer sets designed from the rDNA internal transcribed spacer (ITS) sequences of F. torreyae and F. umbrinellus was developed. The optimal conditions for simultaneous detection of the two pathogens were investigated. The best results were obtained at a reaction temperature of 65°C and a primer ratio of 1:1.5 (F. torreyae : F. umbrinellus). Fluorescently labelled mLAMP amplicons were precipitated using polyethyleneimine. As a result, multiplex detection was enabled by the fluorescent colour of precipitate under ultraviolet light. The detection limit of mLAMP was 100 fg of genomic DNA, which was 10 times more sensitive than the polymerase chain reaction (PCR) method. The mLAMP assay was applied to Japanese pear trunk samples from Aichi Prefecture, Japan, and the results were compared with those obtained using PCR. As a result, mLAMP was observed to be effective for the specific detection of F. torreyae or F. umbrinellus.