Synchronous Detection of DNA/RNA of Four Shrimp Viruses by Real-time Fluorescence Quantitative RT-PCR

[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.

一种简便的同步提取烟草叶片DNA和总RNA的方法

以野生型和转几丁质酶烟草株系叶片为材料,采用改进的CTAB法对其DNA和总RNA进行同步提取和DNA中RNA的消化以及总RNA中DNA的消化,并分别进行了电泳检测、含量测定和相应的PCR和RT-PCR检测。结果表明:用该法提取的DNA条带清晰、无降解,所提取的DNA浓度在109.20～245.46μg/mL,OD_(260)/OD_(280)在1.83～1.94。以提取的DNA为模板,PCR能够扩增出清晰的目的条带。RNA具有5.8SrRNA、18S rRNA和28S rRNA 3条清晰的条带,且无降解。RNA浓度在355.57～570.13μg/mL,OD_(260)/OD_(280)在1.84～2.04,OD_(260)/OD_(230)在2.06～2.33。RNA逆转录成cDNA,经PCR扩增出清晰的看家基因和目的基因条带。

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis

Hepatitis C virus(HCV)infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20%of the total population are infected.Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma.The management of HCV infection should not only be focus on therapy,but also to screen carrier individuals in order to prevent transmission.In the present,molecular detection and quantification of HCV genome by real time polymerase chain reaction(PCR)represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens.However,real time PCR is a complicated approach and of limited distribution.On the other hand,isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care.In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.

对虾病毒DNA/RNA的实时荧光定量RT-PCR同步检测方法研究

为建立同步检测对虾DNA/RNA病毒的实时荧光定量反转录PCR(RT-PCR)方法,提高检验检疫工作效率,探索了一种新的实时荧光定量PCR扩增反应程序。结果显示,应用优化的实时荧光定量RT-PCR检测4种对虾病毒(白斑综合症病毒、传染性皮下及造血器官坏死病毒,桃拉综合征病毒、黄头病毒)DNA/RNA,扩增曲线形态典型,扩增效率理想(E值分别为1.06、1.07、0.92、0.92),标准曲线线性良好(r=1),重复性一致(标准差0.05～0.46,变异系数0.26%～1.62%),灵敏度高,且与实时荧光定量PCR相比差异不显著(平均Ct值误差0.04～0.40,t值0.53～2.50,P>0.05),检测时间约1h。优化的荧光定量RT-PCR可以用于4种对虾病毒DNA/RNA的快速定量检测。

用mRNA差异显示法分离大鼠吗啡依赖相关基因

目的：分离并克隆大鼠吗啡依赖相关基因。方法：ＳＤ大鼠用递增剂量的吗啡作皮下注射，形成吗啡依赖的动物模型。分离鼠脑的中脑导水管周围灰质、伏核、纹状体等核团，提取总ＲＮＡ，用ｍＲＮＡ差异显示（ｄｉｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙＰＣＲ，ＤＤＰＣＲ）的方法，筛选吗啡依赖大鼠特异表达产物的ｃＤＮＡ片段。用３组锚定引物（Ｔ１２ＭＮ，Ｍ＝Ａ，Ｇ，Ｔ或Ｃ，Ｎ＝Ａ，Ｃ或Ｇ）和６组随机引物（ＡＰ１～ＡＰ６）作不同组合进行ＰＣＲ扩增。结果：获得了８０余个差异表达产物的ｃＤＮＡ片段，经斑点杂交初步筛选，克隆了１０个阳性片段，选取４个克隆进行序列分析，获得了４个ｃＤＮＡ片段。结论：通过ＢＬＡＳＴ数据库序列比较，现已确认获得了４个吗啡依赖表达的新基因片段。

一种用于基因芯片可靠的双轮RNA线性扩增技术

DNA微阵列能在一次实验中检测成千上万个基因的表达情况,有助于阐明疾病发生的分子机制及发现新的诊治靶标.但常规方法需要大量RNA,因而基于T7RNA线性扩增技术逐渐成为微阵列表达谱实验中最常用的探针制备方法.本方法将实验步骤进一步改进,增加额外的一轮体外转录,并结合Klenow酶标记技术来制备cDNA靶标和寡核苷酸芯片杂交.从纳克量级的总RNA起始,本方法和常规的RNA单轮线性扩增法相比,仍然准确地保留了约70%的基因表达信息.同一RNA样本的自身比较实验及重复实验结果也显示,该方法具有较高的可靠性和重复性.RNA双轮体外扩增法需要的起始RNA相对于常规的单轮扩增法减少了很多(10ng甚至更少),因而非常适合分析那些只能提供微量RNA的样本.

DNA-directed assembly of copper nanoblocks with inbuilt fluorescent and electrochemical properties： Application in simultaneous amplification-free analysis of multiple RNA species

The intrinsic affinity of DNA molecules toward metallic ions can drive the specific formation of copper nanostructures within the nucleic acid helix structure in a sequence-dependent manner. The resultant nanostructures have interesting fluorescent and electrochemical properties, which are attractive for novel biosensing applications. However, the potential of using DNA-templated nano- structures for precision disease diagnosis remains unexplored. Particularly, DNA- templated nanostructures show high potential for the universal amplification-free detection of different RNA biomarker species. Because of their low cellular levels and differing species-dependent length and sequence features, simultaneous detection of different messenger RNAs, microRNAs, and long non-coding RNAs species with a single technique is challenging. Here, we report a contemporary technique for facile in situ assembly of DNA-templated copper nanoblocks （CuNBs） on various RNA species targets after hybridization-based magnetic isolation. Our approach circumvents the typical limitations associated with amplification and labeling procedures of current RNA assays. The synthesized CuNBs enabled amplification-free fM-level RNA detection with flexible fluorescence or electrochemical readouts. Furthermore, our nanosensing technique displays potential for clinical application, as demonstrated by non-invasive analysis of three diagnostic RNA biomarkers from a cohort of 10 prostate cancer patient urinary samples with 100%-concordance （quantitative reverse transcription- polymerase chain reaction （PCR） validation）. The good analytical performance and versatility of our method may be useful in both diagnostics and research fields.

Comparison of Molecular Biological Techniques (RAPD, ARDRA and SSrRNA Gene Sequencing) on the Phylogenetic Relationships of Lower Taxa within Spirotrichous Ciliates

The evolutionary relationships of seven spirotrichous ciliates (3 stichotrichs: Oxytricha saltans, O. Ferruginea, Stylonychia mytilus; 4 hypotrichs: Uronychia transfuga, Diophrys appendiculata, Aspidisca steini, Euplotes vannus ) inferred from the SSrRNA (small subunit rRNA) gene sequences and the polymorphic patterns of RAPD (random amplified polymorphic DNA) and ARDRA (amplified ribosomal DNA restriction analyses) fingerprinting are constructed. Compared with that of morphometric characters, the dendrograms from SSrRNA gene using three different calculation methods (distance matrix, maximum parsimony, and UPGMA) agree with the morphological division into two clades, Oxytricha Stylonychia and Uronychia Diophrys Aspidisca Euplotes, though the branching orders within the hypotrichous ciliates are slightly different from morphometric analyses.

病原体RNA和DNA同步聚合酶连反应的方法建立与应用

目的:建立一种可同步检测病原体RNA或DNA的荧光定量RT-PCR方法,以提高突发公共卫生快速检测能力。方法:通过与经典荧光定量PCR的比对来验证新建立的方法能否同步扩增RNA和DNA以及对DNA的扩增效果。结果:新建立的方法可同步扩增RNA和DNA,扩增所需时间为35分钟,扩增曲线呈典型的"S"型,扩增效率E值均在理想值范围(0.916、0.923)、均在理想值范围(相关系数均可达到"1"),重复性良好(s均≤0.34、cv均≤1.13%),扩增灵敏度相同(平均ct值误差分布在0.02～0.25之间,t值分布在0.17～2.50之间,P均>0.05)。结论:新建立的方法可同步检测6种不同核酸类型的病原体,检测时间明显缩短,检测效果与经典荧光定量PCR相同。

Engineering Multipedal DNA Walker on Paper for Sensitive Electrochemical Detection of Plant MicroRNA

MicroRNAs (miRNAs) are vital regulators in both plants and animals. Therefore, it is highly desirable to develop portable and user-friendly biosensors for convenient and sensitive detection of miRNAs. Herein, a novel paper-based electrochemical biosensor was intelligently engineered for the detection of plant miRNA based on a smart DNA walking machine and λ-exonuclease (λ-Exo)-assisted target recycling amplification. Using TaMIR5086 as a target plant miRNA, the presence of TaMIR5086 could initiate the target recycling process and activate the DNA walker to move along the track on the paper. Then, numerous electroactive molecules-labeled single-strand DNA (ssDNA) would be released and adsorbed onto the surface of screen-printed electrode, generating a remarkably increased electrochemical signal. Benefitting from the dual amplification, the developed biosensor exhibits excellent analytical performance toward TaMIR5086 with a detection limit down to 0.37 pmol/L. Furthermore, the paper-based biosensor could be applied for the analysis of target miRNA in complex biological samples, which found great potential in the fields of miRNA analysis and plant biology research.

核酸检测技术在临沂市无偿献血人群血液筛查中的应用分析

目的:探讨核酸检测技术在无偿献血人群血液筛查中的应用效果。方法:2015年4月~2016年12月,共122261名无偿献血者进行了血液采集和检测,并对两种血清学检测试剂检测结果呈单阳性和阴性的献血者样本进行核酸检测,对核酸检测阳性献血者进行随访。结果:121978份进行核酸筛查的标本中,共检测出97份HBV DNA阳性和1份HIV1/2 RNA阳性,检测阳性率0.08%,无HCV RNA阳性检出;对15名HBV DNA阳性献血者随访复检12份(80%)阳性;1名HIV1/2 RNA阳性献血者复检阳性,确证感染。结论:单采用酶免技术进行血液筛查存在较高HBV、HIV1/-2 RNA输血感染风险;核酸检测可缩短血液病毒检测“窗口期”,对艾滋病病毒早期检出及降低隐匿性乙型肝炎病毒输血传播风险起到一定作用。

Express and sensitive detection of multiple miRNAs via DNA cascade reactors functionalized photonic crystal array

Array based detection techniques with fluorescence signal reading is a powerful tool for multiple targets analysis. However,when applied fluorescence array for micro RNA detection, time-consuming multi-steps surface signal amplification is usually required due to the low abundance of micro RNA in total RNA expressions, which impairs detection efficiency and limits its application in point of care test(POCT) manner. Herein, DNA cascade reactors(DCRs) functionalized photonic crystal(PC)array was fabricated for express and sensitive detections of mi RNA-21 and mi RNA-155. DCRs were assembled by interval conjugation of self-quenched hairpin DNA probes to single strand DNA nanowire synthesized by rolling circle amplification,which generated cascade DNA hybridization reactions in response to target mi RNAwith instant fluorescence recovery signal. PC array patterns with multi-structure colors further amplified fluorescence with their respective photonic bandgaps(PBGs)matching with the emission peaks of fluorescence molecules labelled on DCRs. The as-prepared DCRs functionalized PC array demonstrated express and sensitive simultaneous detections of mi RNA-21 and mi RNA-155 with hundreds f M detection limits only in 15 min, and was successfully applied in fast quantifications of low abundance mi RNAs from cell lysates and spiked mi RNAs from human serum, which would hold great potential for disease diagnosis and therapeutic effect monitoring with a POCT manner.

Enzyme-free and multiplexed micro RNA detection using micro RNA-initiated DNA molecular motor

In this work,we have developed a sensitive,simple,and enzyme-free assay for detection of micro RNAs(mi RNAs)by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains.In the presence of mi RNA target,it can hybridize with one of the stem-loop DNA to open the stem and to produce a mi RNA/DNA hybrid and a single strand(ss)DNA,the ss DNA will in turn hybridize with another stem-loop DNA and finally form a double strand(ds)DNA to release the mi RNA.One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence.The formation of ds DNA can produced specific fluorescence signal for mi RNA detection.The released mi RNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor,which results in great fluorescence amplification.With the efficient signal amplification,as low as 1 pmol/L mi RNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained.Moreover,by designing different stem-loop DNAs specific to different mi RNA targets and labeling them with different fluorophores,multiplexed mi RNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum(SFS)technique.

基于多重置换扩增技术的RNA病毒基因组扩增方法研究

为了便于新发或罕见病毒性传染病的筛查检测,本研究利用多重置换扩增技术,以负链RNA病毒—发热伴血小板减少综合征病毒和正链RNA病毒—登革病毒为模拟样本探索临床样本中RNA病毒基因组非特异性扩增方法。研究中通过梯度稀释的RNA病毒模拟样本中可能存在的不同丰度的病原体,样本核酸依次加工成单链cDNA、双链cDNA、T4DNA连接酶处理后的双链cDNA以及添加外源辅助RNA后合成并连接的双链cDNA形式,然后进行Phi29DNA聚合酶等温扩增,使用荧光定量PCR方法比较各种方法对RNA病毒核酸扩增的影响。结果显示,对于不同类型的RNA病毒模拟标本,多重置换扩增对于单链及双链cDNA的扩增效果有限,而双链cDNA经DNA连接酶处理后的扩增能达到6×103倍;在cDNA合成过程中加入外源辅助RNA,模拟样本中病毒基因组的扩增可达2×105倍,尤其是对含有低丰度病原体的模拟样本扩增效果的改善更为明显。本研究摸索建立了基于多重置换扩增技术的RNA病毒基因组扩增方法,能够对样本中低丰度RNA病毒基因组实现有效扩增,可满足开展多种病原体筛查检测的需求。

环媒恒温扩增技术在病毒感染检测中的应用

环媒恒温扩增法（loop mediated isothermal amplification，LAMP）是一种新型的恒温核酸扩增技术，它利用了2对特异引物和一种有链取代活性的DNA聚合酶，通过使扩增产物形成单链环结构实现核酸的恒温扩增。LAMP自身的一系列特性使其可以达到对特定核酸片段高效、特异的扩增。由于反应结果可以通过测定反应副产物—焦磷酸镁沉淀引起的浊度来判断，因此使其成为一种十分有效的分子检测手段。

男性淋病奈瑟球菌3种不同检测方法的对比研究

目的对比研究3种检测方法在淋病奈瑟球菌诊断中的临床应用价值。方法收集370例疑似淋病奈瑟菌感染男性患者的尿道分泌物和尿液标本,分别进行以下3种方法检测:普通培养法、DNA实时荧光定量法(RT-PCR)及RNA实时荧光恒温扩增检测(SAT)法。以普通培养法作为对照组,对比3组检测方法对淋病奈瑟菌的阳性率以及检测淋球菌的符合度情况。结果普通培养法、RT-PCR、SAT法阳性检出率分别为24.1%、11.8%、24.8%;普通培养法与SAT法淋病奈瑟菌阳性率均高于RT-PCR法,差异有统计学意义(P<0.05);普通培养法与SAT法淋病奈瑟球菌阳性率差异无统计学意义(P>0.05)。结论在临床工作中对男性淋病奈瑟球菌进行诊断时,建议选择2种以上的检测方法以提高阳性检出率;SAT法阳性反映活细菌数,可以更好地指导临床抗生素合理使用。