DNA电化学生物传感器检测病原微生物的应用研究进展

病原微生物的快速检测对于疫情防控具有重要意义。与传统检测方法相比,DNA电化学生物传感器在检测度、灵敏度、检测成本与便携性等方面有诸多优势。该文综述了DNA电化学生物传感器的工作原理及其在病原体检测中的应用,重点阐述了核酸四面体结构探针和新型纳米材料在DNA电化学生物传感器中的最新进展,以及DNA电化学生物传感器检测技术在病原体现场快速检测中面临的挑战和发展趋势。     

免疫电化学生物传感器的研制及其在炭疽芽孢检测中的应用

免疫电化学生物传感器是诸多生物传感器中的一种,它利用抗原与抗体反应的高亲和力和特异性结合的特点,以抗原或抗体作为敏感材料,并用电化学检测系统作为换能器,从而实现对致病微生物等的检测.目前,国内外关于生物传感器研究的报道很多,但真正商品化传感器有限.我们采用免疫电化学的方法进行微生物检测器的研制.其基本原理为:首先,在固相载体上固定化抗体,然后通过夹心法结合待检测抗原及酶标抗体;由标记酶催化相应的化学反应,反应产物在电化学电极上再发生氧化还原反应,从而形成可以检测的电流信号.我们对两种固相载体的抗体固定化进行了研究,一种是表面带有羧基或胺基的微孔尼龙膜,通过碳二亚胺活化或戊二醛法共价偶联抗体.     

基于CRISPR均相电化学生物传感器检测SARS-CoV-2和HIV-1方法的建立

目的 建立基于CRISPR-Cas13a系统的均相电化学生物传感器并用于病原微生物的核酸检测。方法通过逆转录-重组酶介导链替换核酸扩增技术（RT-RAA）对目标核酸进行扩增,经过体外转录后,激活Cas13a蛋白高效切割单链RNA的活性并对标记有亚甲基蓝（MB）的报告RNA进行剪切,构建CRISPR均相电化学生物传感器;通过遴选报告RNA上寡核苷酸的长度优化该传感器;利用该传感器检测新型冠状病毒（SARS-CoV-2）RNA和Ⅰ型人类免疫缺陷病毒（HIV-1）RNA,并对其检测性能进行评价。结果 建立了一种基于CRISPR-Cas13a系统和RT-RAA等温扩增技术的均相电化学生物传感器,利该方法使用相对应的扩增引物和CRISPR RNA(crRNA)后能稳定检测出低至102拷贝/μl的SARS-CoV-2 RNA和HIV-1 RNA,并与其他多种病原体未发生交叉反应。结论该基于CRISPR-Cas13a系统的均相电化学生物传感器具有制备简单、灵敏度高、特异性好和通用性强等优点,为病原微生物的现场快速检测提供了新的可行方案。     

氨茶碱分子印迹电化学传感器的研制及应用

目的：建立一种可用于早产儿氨茶碱血液浓度的检测方法。方法以氨茶碱为模板分子,吡咯为功能单体,在0．2 mol／L的HAc-NaAc缓冲液（pH 4．0）中,通过电聚合方法在玻碳电极表面聚合形成氨茶碱分子印迹传感膜;利用三维激光扫描显微镜、差分脉冲伏安法（ DPV）和电化学交流阻抗法（ EIS）对分子印迹传感膜的表面形貌及性能进行表征;在5 mmol／L K3[Fe（CN）6]－0．1 mol／L KCl溶液中,采用方波伏安法（SWV）考察了分子印迹膜的聚合扫描圈数和孵育时间对传感器响应的影响。结果在优化实验参数下,传感器SWV峰电流差值与氨茶碱的浓度负对数在1×10－7～1×10－3mol／L范围呈现良好的线性关系,检出限为0．5×10－8mol／L（S／N＝3）,加标回收率为92．2％～101．4％;构建的氨茶碱分子印迹电化学传感器具有良好的选择性、稳定性和重现性。结论所构建的氨茶碱分子印迹电化学传感器有望用于临床上早产儿血液氨茶碱分子浓度的快速及准确检测。     

基于智能手机的生物传感器及其在即时检测中的应用进展

日常健康管理对即时检测(POCT)的需求不断增加,而生物传感器作为一类高灵敏、微型化的生物标志物检测仪器,在POCT中具有巨大的应用潜能。近年来,智能手机的普及和生物传感器技术的进步促进了基于智能手机的生物传感器的发展,这类生物传感器具有便携、快速、低成本、易操作等优点,可以满足POCT对生物传感器日益严格的要求。从基于智能手机的光学生物传感器技术(包括显微镜成像技术、比色技术、荧光技术、生物发光技术和表面等离子共振SPR技术)和电化学生物传感器技术(包括电流法、电位法以及阻抗法)两个方面,介绍了基于智能手机的生物传感器及其在POCT研究中的应用,并关注了其在航天医学领域的应用潜力。     

辣根过氧化酶直接标记人巨细胞病毒DNA探针的制备和临床应用

用活化或根过氧化酶复合物直接标记人巨细胞病毒（HCMV）DNA特异片段制备酶标基因探针。与靶DNA杂交后酶催化检测系统中相应的发光剂，再经增强剂作用将光信号放大，杂交信号通过X光自显影。经决缝杂交证明该法标记的基因探针可检测到0．2Pg的同源DNA，且仅与HCMVDNA杂交，与HSV-1、EBV及正常人DNA不杂交。用此探针检测42例巨细胞包涵体（CID）患儿和20例正常同龄儿童尿中HCMVDNA，阳性率分别为550％和35％。与病毒分离相比较，杂交法敏感性为92．3％，符合率为87．1％，临床标本检出率高于病毒分离法，表明用HRP直接标记基因深外经化学发光自显影检测HCMVk安全、快速、敏感和特异的方法，适用于临床诊断和研究。     

以PBP2a为靶点应用生物传感器技术筛选拮抗MRSA中药

目的 应用生物传感器技术从传统抗炎中药中筛选具有拮抗细菌PBP2a活性的中药并进行活性物质含量的排序.方法 将可溶性PBP2a包被于生物传感器的生物素化样品池以建立靶点,测定115种中药水煎液去鞣质后与PBP2a的亲和力;选择亲和力较高的25种药物水提液和醇提液与定量PBP2a(50ng·ml-1)37℃混合孵育30min,再测定其与PBP2a的亲和力,以评价中药里活性物质的含量.结果 115种中药中,毛冬青、金荞麦、雷公藤等25种中药与PBP2a具有较高亲和力(大于100RU);黄药子、五倍子、半枝莲、山豆根、黄连、草果、核桃根等7种中药中与PBP2a特异性结合的活性物质含量较高.结论 应用生物传感器跟踪技术筛选具有结合PBP2a作用的中药具有可行性,筛选出的7种中药均含有非鞣质类能与PBP2a发生特异性结合作用的活性物质,该7种中药能不同程度抑制MRSA的生长.     

应用地高辛配基标记cDNA探针检测肾综合征出血热病人尸检组织中病毒RNA

应用地高辛配基标记的流行性出血热汉坦病毒RNA M和S片段cDNA探针对因出血热死亡的病人15种尸体解剖组织的51例石蜡切片进行病毒RNA原位杂交检测.结果显示,51例尸检组织中,病毒RNA M片段阳性检出率为70.59%(36/51),S片段为76.47%(39/51).病毒RNA主要存在于各种上皮组织胞浆中,呈包涵体样或弥漫分布.提示,汉坦病毒是一种泛嗜性病毒,主要侵害人体多种上皮组织,其侵害能力因地区不同亦有差异.用原位杂交法在尸检组织中直接检测病毒RNA是一种敏感而特异的方法,优于免疫组化法.     

聚合酶链反应(PCR)检测水中钩体DNA方法的建立和初步应用

目的:为了建立一个检测水中钩体DNA的技术方法。方法:本研究采用了根据1993年C.Gravekamp设计的引物G_1G_2序列自行合成的引物G_1G_2建立了检测致病钩体DNA的PCR方法。运用此方法并结合“优筛法”对采自云南河口、蒙自等地区108份水样进行检测,并与分离培养后经中国药品生物制品检定所鉴定结果进行了比较。结果:两组方法检测所有水样呈阴性,其精确度和一致性达100％,但分离培养方法漏检率为8.3％(9/108)。结论:建立了PCR检测水中钩体DNA方法。此方法简单、快速、准确,适用于一般实验室及大规模水样钩体DNA的检测,值得推广应用。     

EBOV三聚体糖蛋白在新型糖基工程酵母中的表达与纯化

目的 用新型糖基工程酵母表达并纯化得到具有类似哺乳动物细胞糖基化修饰的埃博拉病毒三聚体糖蛋白(EBOV-GP),为其亚单位疫苗研究提供基础.方法 人工合成EBOV-GP基因,将编码全长EBOV-GP基因和编码缺失黏蛋白样域(MLD)与穿膜区的EBOV-GPΔMLDΔTM基因分别克隆到pPICZ-αA载体上,电转化糖基工程酵母,与在HEK-293T细胞中表达的EBOV-GP进行比较,利用PNGaseF和EndoH酶切分析其糖基化修饰,利用亲和层析与离子交换层析纯化目的蛋白,蛋白质N端测序分析其在蛋白翻译修饰过程中是否正确切除了信号肽,同时利用凝胶柱分析是否能形成三聚体结构.结果 PNGaseF酶切结果显示,用糖基工程酵母和HEK-293T细胞表达的EBOV-GP糖蛋白相对分子质量大小与N-糖基化程度均一致,EndoH酶切显示EBOV-GPΔMLDΔTM的N-糖基化修饰是非高甘露糖形式的复杂型糖基化修饰;经三步纯化得到的目的蛋白,N端测序显示为GP蛋白成熟肽序列,其自身信号肽被正确切除;凝胶柱分析显示纯化得到的目的蛋白以三聚体形式存在.结论 用糖基工程酵母表达制备了具有复杂型糖基化修饰的EBOV-GP.     

New stutter ratio distribution for DNA mixture interpretation based on a continuous model

In forensic science, DNA mixture interpretation is traditionally based on a binary model, which does not account for peak-height information in DNA profiles. In recent years, some countries have adopted a continuous model in which peak heights are used and stochastic effects are considered to enable rigorous calculation of likelihood ratios. However, this model requires certain biological parameters which affect the expected allelic and stutter peak heights. In this paper, we focused on estimating the distribution of the stutter ratio (SR) in 15 short tandem repeat loci in relation to the allele repeat number. We estimated the SR values of 234 single-source DNA samples by using a commercially available kit. In all loci except for D8S1179, D21S11, and D2S1338, a simple log-normal distribution model was fitted to the variability of SR. For D21S11, we developed a new distribution model in which distinct log-normal distributions between complete and incomplete repeat units are used (a separate log-normal distribution model). For D8S1179 and D2S1338, we developed another new distribution model that mixes two log-normal distributions to explain two types of repeat structures appearing within the same number of allele repeats. These two models were fitted to the observed SR values more accurately than the simple log-normal distribution model. We expected these new SR models to be applied to DNA mixture interpretation based on a continuous model.     

乙型肝炎e抗原、前S1抗原、前S2抗原与乙型肝炎病毒DNA关系探讨

目的 探讨乙型肝炎e抗原(HBeAg)、前S1抗原(Pre-S1)、前S2抗原(Pre-S2)与其病毒DNA(HBVDNA)的关系。方法 收集268例乙型肝炎患者血标本,以荧光定量PCR法检测HBVDNA,时间分辨荧光免疫分析法检测HBeAg,酶联免疫吸附法检测Pre-S1、Pre-S2。结果 HBVDNA阳性组HBeAg、Pre-S1、Pre-S2阳性率分别为48.2%、76.4%、100%,HBVDNA阴性组分别为1.6%、36.3%、32.3%,两组间每指标阳性率比较,差异均有显著性(均P<0.01)。结论 HBeAg、Pre-S1、Pre-S2与HBVDNA关系密切,以HBVDNA阳性为参照标准,反映病毒复制的敏感性Pre-S2最高,其次为Pre-S1,HBeAg较低;Pre-S1、Pre-S2可作为HBVDNA的补充指标。     

增强子Ⅰ对乙型肝炎病毒DNA疫苗免疫应答的影响

目的 研究乙型肝炎病毒（HBV）自身增强子I（enhancerI，ENHI）对HBV DNA疫苗免疫应答的影响。方法 采用PCR法以HBVadr亚型全基因DNA序列为模板分别扩增表面抗原（HBsAg）和HBsA-ENHI基因片段，重组到载体VR1012中，构建两种HBV DNA疫苗，转染CADS-7细胞及HepG2细胞并免疫BALB／c小鼠。采用蛋白印迹、ELISA、ELISPOT等方法检测其在COS-7和HepG2细胞内的表达及小鼠的体液及细胞免疫应答效果。结果 转染的HepG2和COS-7细胞均表达HBsAg;连接ENHI的HBV DNA疫苗转染HepG2细胞后HBsAg表达量明显升高，两种疫苗转染COS-7细胞表达HBsAg无明显差异；免疫小鼠后第2周产生HBsAb及HBsAg特异性细胞毒T淋巴细胞（CTL），两种疫苗免疫产生的HBsAb及HBsAg特异性CTL无明显差异。结论ENHI可使HBV DNA疫苗转染HepG2细胞表达HBsAg明显增加，对转染COS-7细胞表达HBsAg及接种BALB／C小鼠引起的免疫应答无明显影响。     

输血传播病毒-DNA聚合酶链方法的建立及应用

目的：建立输血传播病毒（ＴＴＶ）－ＤＮＡ聚合酶链反应方法并应用于新疆地区不同人群ＴＴＶ感染的检测。方法：根据已报道的ＴＴＶ基因痛列（ＤＤＢＪ序列号：ＡＢ００８３９４）,通过引物设计软件ＳＱＮＣＥ和ＯＬＩＧＯ在其ＯＲＦ１区设计一对ＰＣＲ引物,扩增产生一个３１５ｂｐ的扩增片段,产物经ＢｇⅠⅡ酶切分别产生一个２１９ｂｐ和９６ｂｐ的酶切片段。结果：用建立的ＰＣＲ方法在１５例维存尔族转氨酶升高的非甲－非庚型     

A mixture detection method based on separate amplification using primer specific alleles of INDELs-a study based on two person's DNA mixture

Samples containing unbalanced DNA mixtures from individuals often occur in forensic DNA examination and clinical detection. Because of the PCR amplification bias, the minor contributor DNA is often masked by the major contributor DNA when using traditional STR or SNP typing techniques. Here we propose a method based in allele-specific Insertion/Deletion (INDEL) genotyping to detect DNA mixtures in forensic samples. Fourteen INDELs were surveyed in the Chinese Han population of Shanxi Province. The INDELs were amplified using two separate primer-specific reactions by real-time PCR. The difference Ct value of the 2 reactions (D-value) were used for determination of the single source DNA. INDELs types and further confirmed by electrophoresis separation. The minor allele frequency (MAF) was above 0.2 in 10 INDELs. The detection limit was 0.3125 ng–1.25 ng template DNA for real-time PCR in all 14 INDEL markers. For single source 10 ng DNA, the average D-value was 0.31 ± 0.14 for LS type, 6.96 ± 1.05 for LL type and 7.20 ± 1.09 for SS type. For the series of simulated DNA mixture, the Ct value varied between the ranges of single source DNA, depending on their INDEL typing and mixture ratios. This method can detect the specific allele of the minor DNA contributor as little as 1:50 in rs397782455 and rs397696936; 1:100 in rs397832665, rs397822382 and rs397897230; the detection limit of the minor DNA contributor was as little as 1:500–1:1000 in the rest INDEL markers, a much higher sensitivity compared with traditional STR typing. The D-value variation depended on the alternation of dilution ratio and INDEL types. When the dilution was 1:1000, the maximum and minimum D-values were 8.84 ± 0.11 in rs397897230 and 4.27 ± 0.19 in rs397897239 for LL and SS type mixture, the maximum and minimum D-values were 9.32 ± 0.54 in rs397897230 and 4.38 ± 0.26 in rs 397897239 for LL(SS) and LS type mixture, separately. Any D-value between 0.86 and 5.11 in the 14 INDELs indicated the presence of mixture. The separate amplification strategy based on real-time PCR provides a promising and convenient method for detection of unbalanced DNA mixture for Chinese Han population.     

Trace DNA evidence dynamics: An investigation into the deposition and persistence of directly- and indirectly-transferred DNA on regularly-used knives

Empirical data on the transfer and persistence of trace DNA are crucial to the evaluation of forensic DNA evidence. This evaluation can be complicated by the occurrence of indirect DNA transfer; the possibility of which is well established, but research into such transfer is often focussed on unrealistic situations, e.g. handling of DNA-free items after participants have shaken hands for 1–2 min. To simulate more realistic scenarios, this study investigated the deposition and persistence of both directly- and indirectly-transferred DNA on knives that had been artificially set up as ‘regularly-used’. Each knife was handled in a prescribed manner by a specific participant over two consecutive days to simulate regular use. Each participant then shook hands for 10 s with a fellow volunteer and immediately stabbed one of their knives into a foam block repeatedly for 60 s. DNA was recovered by mini-taping from triplicate sets of knife handles from four pairings of volunteers after regular use, and at one hour, one day and one week after the handshaking and stabbing events.Total amounts of DNA recovered from the knives, regularly used by a single person, varied among individuals; one volunteer consistently deposited significantly greater amounts than the others, whilst another volunteer did not always leave complete profiles. DNA attributed to the regular user persisted for at least a week, declining with increasing time between DNA deposition and recovery. Non-donor DNA was co-deposited at <5% of the profiles recovered, except for one volunteer, who consistently left DNA from their romantic partner on their knives at ∼25% and ∼11% of the profiles before and after the handshaking and stabbing events, respectively. In three pairings of volunteers, after the handshaking and stabbing events, alleles that could be attributed to the respective handshakers’ profiles were detected as partial minor profiles, equating to ∼10% of the profiles recovered. For the fourth pairing of volunteers, only complete single-source DNA profiles matching the regular user’s profile were recovered. However, it is important to note that, when indirectly-transferred handshaker DNA was detected, it declined with increasing time between DNA deposition and recovery.These data provide an initial insight into the detection and persistence of directly- and indirectly-transferred DNA that extend the data already available on forensic DNA transfer. The results herein suggest that the sooner an item is sampled after an offence has occurred, the greater the chance of recovering indirectly-transferred DNA, which has implications for forensic reconstructions.     

Methods enhancement for improved recovery of human DNA from forensic blood samples on different fabrics using the DNA IQ System

DNA typing of biological samples can be a challenging task due to the presence of small amounts of DNA and/or a high content of PCR inhibitors. Our aim is to develop a protocol to recover DNA suitable for DNA typing from blood-stained fabrics based on the DNA IQ System. Blood stains on different fabrics were buried in different types of soil and left for 1–7 days. The samples were then recovered and DNA was extracted with a modified DNA IQ System protocol, involving a modification in the preparation of the Wash Buffer. The samples extracted with the modified protocol showed a higher amount of recovered DNA compared with the recommended protocol. The removal of isopropanol from the Wash Buffer composition contributes to a higher amount of recovered DNA.     

Development of a dual test procedure for DNA typing and methamphetamine detection using a trace amount of stimulant-containing blood

Investigation of drug-related crimes, such as violation of the Stimulant Drug Control Law, requires identifying the used drug (mainly stimulant drugs, methamphetamine hydrochloride) from a drug solution and the DNA type of the drug user from a trace of blood left in the syringe used to inject the drug. In current standard test procedures, DNA typing and methamphetamine detection are performed as independent tests that use two separate portions of a precious sample. The sample can be entirely used up by either analysis. Therefore, we developed a new procedure involving partial lysis of a stimulant-containing blood sample followed by separation of the lysate into a precipitate for DNA typing and a liquid-phase fraction for methamphetamine detection. The method enables these two tests to be run in parallel using a single portion of sample. Samples were prepared by adding methamphetamine hydrochloride water solution to blood. Samples were lysed with Proteinase K in PBS at 56 °C for 20 min, cooled at −20 °C after adding methanol, and then centrifuged at 15,000 rpm. Based on the biopolymer-precipitating ability of alcohol, the precipitate was used for DNA typing and the liquid-phase fraction for methamphetamine detection. For DNA typing, the precipitate was dissolved and DNA was extracted, quantified, and subjected to STR analysis using the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. For methamphetamine detection, the liquid-phase fraction was evaporated with N₂ gas after adding 20 μL acetic acid and passed through an extraction column; the substances captured in the column were eluted with a solvent, derivatized, and quantitatively detected using gas chromatograph/mass spectrometry. This method was simple and could be completed in approximately 2 h. Both DNA typing and methamphetamine detection were possible, which suggests that this method may be valuable for use in criminal investigations.