Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.     

Development of an electrochemical DNA biosensor with a high sensitivity of fM by dendritic gold nanostructure modified electrode

In this paper, dendritic gold nanostructure (DenAu) modified electrode was obtained by direct electrodeposition of planar electrode into 2.8 mM HAuCl(4) and 0.1 M H(2)SO(4) solution under a very negative potential of -1.5 V. Scanning electron microscopy was used to characterize the growth evolution of DenAu with time. The whole DNA biosensor fabrication process based on the DenAu modified electrode was characterized by cyclic voltammetry and electrochemical impedance spectroscopy methods with the use of ferricyanide as an electrochemical redox indicator. The probe DNA immobilization and hybridization with target DNA on the modified electrode could be well distinguished by using methylene blue as an electrochemical hybridization indicator. The DenAu modified electrode could realize an ultra sensitivity of 1 fM toward complementary target DNA and a very wide dynamic detection range (from 1 fM to 1 nM).     

A highly sensitive immuno-PCR assay for detection of H5N1 avian influenza virus

With an aim at detecting the ultra-low concentration of avian influenza virus (AIV), a highly sensitive hybrid assay based on immunology and polymerase chain reaction was developed. The TopYield microtiter plates were coated with ten-fold serial dilutions of H5N1 subtype AIV ranging from 10 EID₅₀ ml⁻¹~10⁻⁴ EID₅₀ ml⁻¹,which was recognized by mouse anti-AIV H5 monoclonal antibody (MAb) that was directly linked with reporter DNA using a heterobifunctional cross-linker. After extensive washing, the reporter DNA including a BamH I-restriction site was released by a specific enzymatic restriction, then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition, MAb-based immuno-PCR (IPCR) method could measure 100 μl of AIV H5N1 with 10⁻⁴ EID₅₀ ml⁻¹.To evaluate the sensitivity of IPCR, the same concentration and volume of AIV H5N1 were detected by conventional RT–PCR and sandwich ELISA. The results showed that IPCR had an approximately 1,000-fold improvement over the conventional ELISA, and a 100-fold enhancement compared with RT–PCR in detection sensitivity. To further evaluate the specificity of IPCR for AIV H5 subtype, the tracheal swab specimens, taken from chickens which were infected with H9N2, and the allantoic fluid from eggs inoculated by AIV H3N2, H7N1, H9N2, were detected by IPCR. To mimic clinical samples, pharyngeal–tracheal swab specimens were collected from healthy chickens and spiked with H5N1, H5N2, H5N3 for analysis by immuno-PCR. The results demonstrated that IPCR was a highly sensitive and specific assay for AIV H5, and could be applied to clinical detection for low amount of AIV H5 subtype. This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.     

Tracking cancer cell proliferation on a CMOS capacitance sensor chip

We report a novel technique for assessing cell proliferation that employs integrated capacitance sensors for monitoring the growth of anchorage-dependent living cells. The sensors measure substrate coupling capacitances of cells cultured on-chip in a standard in vitro environment. The biophysical phenomenon underlying the capacitive behavior of cells is the counterionic polarization around the insulating cell bodies when exposed to weak, low frequency electric fields. The sensors employ charge sharing for mapping sensed capacitance values in the fF range to output voltage signals. The sensor chip has been fabricated in a commercially available 0.5microm, 2-poly 3-metal CMOS technology. We report experimental results demonstrating sensor response to the adhesion of MDA-MB-231 breast cancer cells followed by their proliferation on the chip surface. On-chip capacitance sensing offers a non-invasive, label-free, easy-to-use, miniaturized technique with real-time monitoring capability for tracking cell proliferation in vitro.     

禽流感病毒H7N2血凝素HA1基因在大肠杆菌中的表达

目的　表达H7N2亚型禽流感病毒 (AIV)HA1基因 ,用于感染H7亚型禽流感病毒抗体的检测和HA1蛋白功能研究。方法　采用RT PCR方法对H7N2亚型AIVHA1基因进行扩增 ,将PCR产物克隆于pGEM T Easy载体 ,将该基因插入pGEX 4T 2中构建HA1基因原核表达载体 ,转化BL2 1大肠杆菌后 ,在IPTG诱导下表达HA1蛋白 ,Westernblot鉴定表达HA1蛋白。电洗脱方法纯化表达HA1蛋白 ,建立间接ELISA方法 ,对感染AIVH7、H9、H5亚型AIV阳性血清进行检测。结果　成功克隆H7N2亚型AIV的HA1基因 ,其核苷酸序列长度 96 6bp ,编码 32 2个氨基酸残基。构建HA1基因原核表达载体在大肠杆菌内表达出约 6 1× 10 3的HA1融合蛋白。Westernblot和ELISA方法鉴定表明 :表达HA1蛋白与感染H7亚型AIV鸡血清有反应 ,与H5、H9亚型AIV阳性血清没有反应。结论　本研究在大肠杆菌中成功表达了H7N2亚型AIVHA1基因蛋白 ,具有与感染H7亚型AIV阳性血清反应原性 ,不与H5和H9亚型AIV感染阳性血清发生反应。     

禽流感病毒H5N1亚型HA基因表达及其产物的应用

根据GenBank公布的禽流感病毒H5N1亚型血凝素基因(HA)(GenBank:DQ023145)序列设计一对引物P1、P2,以重组质粒pUC-HA为模板扩增去除信号肽的HA成熟蛋白。PCR产物克隆入pMD18-T载体,经测序发现在967位A突变为T,形成一个终止密码子TAA。在突变位点附近设计两条有21个碱基配对的突变引物P3、P4,采用重叠延伸剪切法(SOE)用A定点替换T碱基,然后将正确的基因片段定向插入到表达载体pET-32a( )中,诱导表达获得正确的表达产物。Western-blot分析表明,表达的重组蛋白能与经BL21(DE3)大肠杆菌菌体裂解液处理的H5亚型禽流感病毒阳性抗血清发生特异性反应。利用纯化的重组HA蛋白初步建立了检测H5亚型禽流感病毒抗体的间接ELISA方法,该方法可以代替传统的血凝与血凝抑制方法用于区分禽流感病毒的血清亚型。本研究为禽流感病毒亚单位疫苗及新型诊断试剂盒的研究奠定了基础。     

Efficient capture of infectious H5 avian influenza virus utilizing magnetic beads coated with anionic polymer

The possible emergence of a pandemic influenza virus from the avian influenza virus (AIV) has become a serious threat. The isolation of viruses will be crucial for further virological analysis and the development of vaccines. However, currently, there is no simple method for facilitating the isolation of infectious AIV. Here, we have developed a simple method of capturing AIV using anionic magnetic beads. The method employed the capture of AIV (H5N1, H5N2, and H5N3) from liquid samples such as allantoic fluid (AF) and cell culture medium (CM) using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydride). After their incubation with AIV-containing samples, the magnetic beads were separated from the supernatant by applying a magnetic field. The absorption of AIV on the beads was confirmed by immunochromatography and an enzyme-linked immunosorbent assay, which indicated the presence of hemagglutinin, neuraminidase, and nucleoprotein of AIV. Furthermore, the infectivity in chicken eggs of AIV captured by magnetic beads was similar to that of the starting materials. The capture of AIV using magnetic beads coated with anionic polymers will contribute to the sufficient recovery of infectious AIV and approach for potential pandemic influenza viruses.     

Two DNA Aptamers against Avian Influenza H9N2 Virus Prevent Viral Infection in Cells

New antiviral therapy for pandemic influenza mediated by the H9N2 avian influenza virus (AIV) is increasingly in demand not only for the poultry industry but also for public health. Aptamers are confirmed to be promising candidates for treatment and prevention of influenza viral infections. Thus, we studied two DNA aptamers, A9 and B4, selected by capillary electrophoresis-based systemic evolution of ligands by exponential enrichment (CE-SELEX) procedure using H9N2 AIV purified haemagglutinin (HA) as target. Both aptamers had whole-virus binding affinity. Also, an enzyme-linked aptamer assay (ELAA) confirmed binding affinity and specificity against other AIV subtypes. Finally, we studied aptamer-inhibitory effects on H9N2 AIV infection in Madin–Darby canine kidney (MDCK) cells and quantified viral load in supernatant and in cell with quantitative PCR (qPCR). Our data provide a foundation for future development of innovative anti-influenza drugs.     

H5N1亚型禽流感病毒血凝素基因的原核表达及间接ELISA方法的初步建立

根据GenBank公布的H5N1亚型禽流感病毒(AIV)血凝素(HA)基因序列设计引物,用PCR方法扩增H5N1亚型禽流感病毒HA1基因, 将该片段定向插入到原核表达载体pET_32a(+)中,构建原核表达载体pET_HA1。阳性质粒转化宿主菌BL21(DE3), 经IPTG诱导, HA1基因获得表达, 重组蛋白以包涵体的形式存在。通过改变IPTG的浓度和诱导时间 , 确定了表达HA1基因的最佳诱导条件: IPTG终浓度为0.8mmol/L,诱导时间为3h。Western blot分析表明表达产物具有良好的免疫学活性。以纯化的表达产物作为诊断抗原建立了检测H5亚型AIV抗体的iHA_ELISA方法。结果表明,抗原的最佳包被浓度为4μg/mL,血清的最佳稀释度为1∶200, 阳性标准初步定为：OD待检血清＞05,且 OD待检血清/OD阴性血清＞2。     

AIV血凝素亚型同步检测基因芯片技术研究

对流感病毒14个血凝素亚型的基因芯片检测技术进行了初步研究。通过RT-PCR克隆禽流感病毒血凝素基因片段,获得重组质粒。从重组质粒扩增大约500bp的DNA片段,浓缩后点到氨基化玻璃载体上,制成芯片。待检病毒样品用TRIzolLS提取RNA,反转录过程中用Cy5标记样品cDNAs。将标记样品与芯片杂交,扫描芯片上待检样品与芯片上捕捉探针的结合位点,杂交信号与预期设想一致。结果显示,DNA芯片技术可以提供一种有效的AIV血凝素亚型鉴别诊断方法。     

一株抗H5亚型禽流感病毒血凝素蛋白单克隆抗体的广谱中和活性

以H5N1禽流感病毒株Ck/HK/Yu22/02作为抗原,应用常规杂交瘤技术和血凝抑制实验筛选出抗H5亚型禽流感病毒血凝素蛋白的单抗8H5,单抗8H5经免疫荧光鉴定具有很好的H5特异性.选择33株2002～2006年不同地域,不同宿主中分离的不同遗传变异亚系的H5N1病毒代表株,对单抗8H5分别进行血凝抑制实验及中和试验分析,结果显示单抗8H5对所有H5亚型病毒均有较强反应,而对非H5亚型标准病毒株均不反应,说明8H5是一株广谱性抗H5特异性中和单抗,并提示单抗8H5的HA识别表位可能是一个相当保守的中和表位.并且单抗8H5双抗夹心系统的初步评价显示了其在诊断应用上的前景.     

Detection of bacterial pathogen DNA using an integrated complementary metal oxide semiconductor microchip system with capillary array electrophoresis

In this paper, we show an integrated complementary metal oxide semiconductor (CMOS)-based microchip system with capillary array electrophoresis (CAE) for the detection of bacterial pathogen amplified by polymerase chain reaction (PCR). In order to demonstrate the efficacy of PCR reaction for the heat-labile toxin producing enterotoxigenic Escherichia coli (E. coli), which causes cholera-like diarrhea, 100 bp DNA ladders were injected along with the PCR product. Poly(vinylpyrrolidone) (PVP) was used as the separation medium and provided separation resolution which was adequate for the identification of PCR product. The miniaturized integrated CMOS microchip system with CAE has excellent advantages over conventional instrumental systems for analysis of bacterial pathogens such as compactness, low cost, high speed, and multiplex capability. Furthermore, the miniaturized integrated CMOS microchip system should be compatible with a variety of microfabricated devices that aim at more rapid and high-throughput analysis.     

禽流感病毒A型和H5亚型RT-PCR检测试剂盒研究

目的　检测和鉴定A型、H5亚型禽流感病毒 (AIV) ,研发一种高效实用的检测手段。方法　根据Ming ShiuhLee报道的文献设计、合成引物 ,采用反转录和PCR一步法对A型、H5亚型禽流感病毒cDNA进行扩增和电泳鉴定 ,组装成禽流感病毒RT PCR试剂盒 ,对H1～ 15亚型AIV参考株、38份AIV国内分离株进行检测试验。结果　建立了A型、H5亚型禽流感病毒RT PCR检测方法 ,并在此基础上组装试剂盒 ,用A型试剂盒检测时 ,全部AIV毒株均为阳性 ,能检测 1 10 2 4血凝单位禽流感病毒 ;用H5亚型试剂盒检测时 ,仅有H5亚型AIV参考株和 19株H5亚型AIV分离株呈阳性 ,其余H1～H4、H6～H15参考株和H7、H9分离株以及 1株H5分株均为阴性 ,能检测1 6 4血凝单位禽流感病毒。 2种试剂盒对实验感染鸡病料检出率均为 10 0 %。结论　研制的AIVA型、H5亚型RT PCR试剂盒具有特异性强、敏感性高、稳定性和重复性好的特点。     

利用基因芯片技术区分禽流感病毒主要亚型

[目的]研制可同时区分AIV的H5、H7、H9血凝素亚型及N1、N2神经氨酸酶亚型的基因诊断芯片.[方法]分别克隆了禽流感病毒的M基因,H5、H7、H9亚型HA基因,N1、N2亚型NA基因以及看家基因GAPDH的重组质粒.以重组质粒为模板,用PCR方法扩增制备探针,纯化后点于氨基修饰的片基上,制备基因芯片.在PCR过程中对待检样品进行标记,然后与芯片杂交,洗涤,扫描并进行结果分析.[结果]结果显示检测探针可特异性的与相应的标记样品进行杂交,呈现较强的杂交信号,且无交叉杂交.同时用RT-PCR、鸡胚接种和基因芯片方法对H1-H15亚型AIV参考毒株、30份人工感染样品、21份现地疑似样品进行检测,结果发现,对人工感染样品芯片检测方法与鸡胚接种和RT-PCR的符合率分别为100%和96%,现地样品符合率为100%.[结论]研究表明该方法可用于同步鉴别部分主要流行的禽流感亚型,是一种有效的新方法.     

禽流感病毒分离株NS基因同源性及等位基因类型分析

目的　克隆测定国内具有代表性的禽流感病毒 (AIV)的非结构 (NS)蛋白基因核苷酸序列 ,分析其同源性和等位基因类型 ,为进一步探索禽流感NS蛋白抗体监测方法奠定基础。方法　经RT PCR扩增了国内 3株H9N2、2株H5N1、2株H7N2亚型AIV分离株的NS蛋白基因 ,并把扩增的基因片段克隆到pGEM T载体中测序 ,将测序结果与GenBank中的核苷酸序列进行同源性比较 ,绘制基因进化树。结果　经测序获得了各AIV分离株NS基因的完整编码序列。同源性分析表明 ,3株H9亚型AIV的NS基因之间的同源性为 96 %～ 98% ;两株H5亚型AIVNS基因同源性为 91 6 % ;两株H7亚型AIV的NS基因同源性为 98 9%。H5和H9亚型分离株的NS基因之间的同源性均高于 90 % ;而H7N2亚型分离株与其它两种亚型分离株的NS基因同源性约为 6 0 %～ 70 %。在AIVNS基因系统发育进化树中 ,H5、H9亚型分离株都处于等位基因A群内 ;3株H9亚型分离株的进化关系较近 ,与香港、广东的部分H5N1病毒株起源相同 ,而 2株H5病毒的NS基因则处于不同分枝内 ;2株H7亚型分离株的NS基因都处于等位基因B群内 ,进化关系较近。结论　这 7株国内AIV分离株的NS基因之间的同源性差异较大 ,约为 6 0 %～ 99% ,且包括A、B两种类型的等位基因     

Detection of Hepatitis B Virus (HBV) DNA at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor

We report Hepatitis B Virus (HBV) DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For this assay, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. For efficient detection of the HBV target DNA using silica nanoparticle-enhanced DNA assay, the size of silica nanoparticles and the dimension of microcantilever were optimized by directly binding the silica nanoparticles through DNA hybridization. In addition, the correlation between the applied nanoparticle concentrations and the resonant frequency shifts of the microcantilever was discussed clearly to validate the quantitative relationship between mass loading and resonant frequency shift.HBV target DNAs of 23.1 fM to 2.31 nM which were obtained from the PCR product were detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer was detected up to the picomolar (pM) level without nanoparticle enhancement and up to the femtomolar (fM) level using a nanoparticle-based signal amplification process. In the above two cases, the resonant frequency shifts were found to be linearly correlated with the concentrations of HBV target DNAs. We believe that this linearity originated mainly from an increase in mass that resulted from binding between the probe DNA and HBV PCR product, and between HBV PCR product and silica nanoparticles for the signal enhancement, even though there is another potential factor such as the spring constant change that may have influenced on the resonant frequency of the microcantilever.     

以减毒沙门氏菌运送的H5亚型禽流感病毒DNA疫苗的构建及其对小鼠的免疫原性

根据GenBank中已发表的H5亚型禽流感病毒HA基因序列,设计一对引物,通过RTPCR扩增鹅源H5亚型高致病力禽流感病毒HA基因,测序确认后,将其克隆入真核表达载体pVAX1和asdpVAX1得到重组表达载体pVAX1HA和asdpVAX1HA。将重组质粒转染P815细胞,经间接免疫荧光试验证实,HA基因在细胞内得到了瞬时表达。进一步将重组质粒转化减毒鼠伤寒沙门氏菌X4550得到两种运送DNA疫苗的重组沙门氏菌X4550（pVAX1HA）和X4550（asdpVAX1HA）,以1×109CFU/只的剂量两次口服免疫BALB/c小鼠,免疫小鼠不仅可以检测到HA特异性的血清抗体应答,而且还能抵抗稳定表达H5亚型禽流感病毒HA基因的P815肥大细胞瘤的攻击,说明该运送DNA疫苗的减毒沙门氏菌系统在体内能够成功释放所携带的质粒,并且能够刺激机体产生保护性免疫应答。     

检测H5亚型禽流感的压电免疫传感器的研究

目的：为研制检测H5亚型禽流感的压电免疫传感器。方法：用巯基丙酸在镀银电极石英晶体自组装巯基丙酸单分子膜再通过N-乙基-N’-（3-二甲氨基）丙基碳化二亚胺盐酸（EDC）和N-羟基琥珀酰亚胺（NHS）偶联抗H5亚型禽流感病毒的特异性单抗构建传感器芯片,建立了可以检测H5亚型禽流感病毒的免疫传感器。结果：结果表明,该法具有较好的特异性,不与H9亚型流感病毒和NDV反应;检测灵敏度达到10—50个EID50。结论：本文结果为检测禽流感病毒免疫传感器的进一步深入研究奠定了基础,这为其它相关病毒的监测提供了一种新途径。     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

Chicken cells sense influenza A virus infection through MDA5 and CARDIF signaling involving LGP2

Avian influenza viruses (AIV) raise worldwide veterinary and public health concerns due to their potential for zoonotic transmission. While infection with highly pathogenic AIV results in high mortality in chickens, this is not necessarily the case in wild birds and ducks. It is known that innate immune factors can contribute to the outcome of infection. In this context, retinoic acid-inducible gene I (RIG-I) is the main cytosolic pattern recognition receptor known for detecting influenza A virus infection in mammalian cells. Chickens, unlike ducks, lack RIG-I, yet chicken cells do produce type I interferon (IFN) in response to AIV infection. Consequently, we sought to identify the cytosolic recognition elements in chicken cells. Chicken mRNA encoding the putative chicken analogs of CARDIF and LGP2 (chCARDIF and chLGP2, respectively) were identified. HT7-tagged chCARDIF was observed to associate with mitochondria in chicken DF-1 fibroblasts. The exogenous expression of chCARDIF, as well as of the caspase activation and recruitment domains (CARDs) of the chicken melanoma differentiation-associated protein 5 (chMDA5), strongly activated the chicken IFN-β (chIFN-β) promoter. The silencing of chMDA5, chCARDIF, and chIRF3 reduced chIFN-β levels induced by AIV, indicating their involvement in AIV sensing. As with mammalian cells, chLGP2 had opposing effects. While overexpression decreased the activation of the chIFN-β promoter, the silencing of endogenous chLGP2 reduced chIFN-β induced by AIV. We finally demonstrate that the chMDA5 signaling pathway is inhibited by the viral nonstructural protein 1. In conclusion, chicken cells, including DF-1 fibroblasts and HD-11 macrophage-like cells, employ chMDA5 for sensing AIV.