Topological conformational changes of human papillomavirus(HPV)DNA bound to an insoluble aluminum salt—A study by low temperature PCR

A low temperature (LoTemp?) polymerase chain reaction (PCR), conducted at cycling temperatures not to exceed 85℃and catalyzed by a novel highly processive HiFi? DNA polymerase with proofreading function, was used to study the topological conformational changes of the human papillomavirus (HPV) L1 gene DNA fragments bound to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant in the quadrivalent HPV vaccine, Gardasil?. L1 gene DNA fragments of HPV-11, HPV-18 and HPV-16 were detected in the AAHS particles by nested PCR, but all were lacking a region that was amplifiable by an MY09 degenerate primer. In addition, a pair of degenerate consensus GP6/MY11 primers was able to amplify a target segment of the HPV-11 L1 gene DNA and the HPV-18 L1 gene DNA bound to the AAHS particles as expected for any HPV DNA in the B-conformation. However, there was no co-amplification of the HPV-16 L1 gene DNA known to coexist in the same samples. The lack of co-amplification was verified by direct DNA sequencing of the PCR amplicons. The companion HPV-16 L1 gene DNA in the same sample required repeated PCRs with a pair of modified non-degenerate GP6/ MY11 primers for detection. This melting profile of the HPV-16 L1 gene DNA was similar to that of the HPV-16 L1 gene DNA recently discovered in the postmortem blood of a young woman who suffered a sudden unexpected death 6 months after Gardasil? vaccination. The findings suggest that the topological conformational changes in the HPV L1 gene DNA residues bound to the AAHS adjuvant may be genotype-related. The special non-B-conformation may prevent the HPV-16 L1 gene DNA from being degraded in the body of the vaccine recipients after in- tramuscular injection.

Effect of Low Temperature Stress on Sweet Potato S-Adenosyl Methionine Synthetase Gene Expression

[Objective] The mRNA expression level changes of S-adenosylmethionine synthetase （SAMS） under low temperature stress was studied. [Method] Total RNA were extracted from leaves, stem and earthnut of sweet potato 0,12,24,48 and 72 h after low temperature treatement, mRNA expression level was analyzed by reverse expression and Real-time PCR technique. [Result] The expression quality of the gene extracted from leaves, stem and earthnut increased and the expression quality reached the peak point 24,72 and 72 h after low temperature treatment respectively. The expression change of earthnut was the biggest. [Conclusion] Low temperature was good for increasing mRNA expression of relevart genes.

Gene Expression and Activities of SOD in Cucumber Seedlings Were Related with Concentrations of Mn^(2+),Cu^(2+),or Zn^(2+) Under Low Temperature Stress

Effects of increasing Mn^2＋, Cu^2＋, or Zn^2＋ on SOD expressions were studied in cucumber seedlings under low temperature stress. Both gene expressions and activities of Cu/Zn-SOD and Mn-SOD in cucumber seedling leaves were induced by increasing Mn^2＋, Cu^2＋, or Zn^2＋ under low temperature stress, especially 48 h afterwards. The activities of Cu/Zn-SOD and Mn-SOD at 0 and 48 h after treatment were in accordance with their gene expression levels, which implied that the transcriptional regulation plays key roles in regulating their activities at the early stage of low temperature stress. Gene expressions of Cu/Zn-SOD and Mn-SOD declined at 96 h, but Cu/Zn-SOD and Mn-SOD activities still remain high, which suggested that Cu/Zn-SOD and Mn-SOD activities might be regulated by other factors after transcription at the later stage of low temperature stress. Therefore, we concluded that the increasing Mn^2＋, Cu^2＋, or Zn^2＋ could increase the capacity of scavenging ROS in cucumber seedlings under low temperature stress by inducing gene expressions of Cu/ Zn-SOD and Mn-SOD, elevating activities of Cu/Zn-SOD, Mn-SOD, or regulating other factors after transcription.

Temperature Stress at Grain Filling Stage Mediates Expression of Three Isoform Genes Encoding Starch Branching Enzymes in Rice Endosperm

An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.

Influence of temperature on the characteristics of aerobic granulation in sequencing batch airlift reactors

Aerobic granular sludge was cultivated in sequencing batch airlift reactors （SBAR） at 25, 30, and 35℃, respectively. The effect of temperature on the granules characteristics was analyzed and the microbial community structures of the granules were probed using scanning electron microscope （SEM） and polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE）. The results showed that 30℃ is optimum for matured granule cultivation, where the granules had a more compact structure, better settling ability and higher bioactivity, the oxygen utilization rate （OUR） reached 1.14 mg O2/（g MLVSS.min） with COD removal rate of 97% and TP removal rate of 75%. The removal efficiency of NH3-N increased from 68.5% to 87.5% along with the temperature increment from 25 to 35℃. The DGGE profiles revealed that the microbial community structure at 25℃ showed the least similarity with those at other temperatures. The sequencing results indicated that the majority of dominant microbes belonged to Actinobacteria and Proteobacterium. Thermomonas sp., Ottowia sp. and Curtobacteriurn ammoniigenes might play important roles at different temperatures, respectively.

Differential Expression of miRNAs in Rice under High Temperature Stress

The growth and development of rice are closely related with temperature. In order to clarify the mechanism of high temperature resistance in riee, in this study, using high temperature-resistant Indian rice cultivar N22 as the experimental material, Osa-rniR159c, Osa-miR159d, Osa-miR159f, Osa-miR164d, Osa- rrdR529b and Osa-miR166h-3p obtained by high-throughput sequencing as target genes, the expression patterns of these genes in young panicles of rice under high temperature stress were analyzed by RNA-tailing and primer-extension RT-PCR, which provided theoretical basis for breeding high temperature-resistant rice eultivars.

一种精准控制管内温度的低成本PCR系统设计

现有商用聚合酶链反应(PolymeraseChainReaction,PCR)仪以金属孔板作为控制对象,为了使管内样本温度维持足够时间,常以延长程序设定时间来对冲管内温度滞后对检测结果的影响,但会导致实验耗时长、管内温度不精确等后果.本文提出一种利用导热金属孔板过冲热量抵消PCR管内温度滞后的方法对管内温度进行修正,设计开发热循环控制系统,并用非洲猪瘟模板进行实验验证.结果表明,PCR管内温度校正后升降温耗时分别缩短了56.7%和28.6%,PCR扩增曲线Ct值提前了2.64±0.48,有效提升了PCR的检测效率及结果可靠性.

384孔PCR仪温度参数校准方法探讨

PCR技术作为一项革命性的分子生物学工具,在基因分析、病原体检测、药物研发、遗传学研究等领域发挥着重要作用。384孔PCR仪作为高通量PCR仪的代表,具备一次性处理多个样本的能力。由于现有的校准规范主要针对96孔,使用384孔时存在校准困难和准确性较差的问题。因此,本文通过对两者进行对比,提出了384孔PCR仪的校准布点方式,并进行试验验证。结果表明:采用该校准布点方式具有有效性。

基于多传感器融合技术的PCR仪温度校准系统

提出了一种基于多传感器融合技术的PCR仪温度校准系统。系统对特征点的温度检测和处理后能给出PCR仪基座的温度均匀性和对称性,同时对采集到的多点温度数据运用多传感器数据融合技术进行处理,得到更接近真实基座温度的融合温度,从而可以得到仪器的温度示值误差,以实现对PCR仪的设定温度进行校准。系统选择LabVIEW作为开发软件,能自动生成检测报告。系统的温度校准不确定度优于0.03℃。实验表明,建立的温度校准系统可以用于目前大多数使用中的PCR仪温度的在线校准。

定量PCR仪热循环系统温度均匀性有限元仿真研究

温度均匀性是衡量定量PCR仪温度控制效果的重要指标,也是决定定量结果准确性的基本因素。通过研究PCR仪热循环系统在热循环过程中的温度场分布情况及影响温度场分布均匀性的各项因素,使用大型有限元仿真软件ABAQUS6.8分别对影响温度场分布的空气对流、导热胶、TEC匹配等因素进行了仿真分析。根据仿真结果,对改善温度均匀性提出了解决方法,分别通过实验和仿真给予验证。结果表明,通过匹配TEC,采取隔热或热补偿的措施,可有效改善96孔样品块的温度均匀性。

两温度梯度多重PCR鉴别牛早期胚胎性别的技术

稳定、可靠和快速的牛胚胎性别鉴定方法在生产应用中具有重要意义。通过两温度梯度PCR方法对牛基因组、克隆胚胎、胚胎样品进行性别鉴别实验研究,建立了稳定、简便、快速的牛早期胚胎性别鉴别两温度梯度PCR方法,鉴定时间仅为57分钟。采用两温度梯度PCR方法对30枚奶牛胚胎进行了早期性别鉴别,并将鉴别的15枚胚胎(11枚为雌性,4枚为雄性)移植到同期处理的15头受体母牛体内。60天后妊娠检查,有7个受体成功受孕,5头受体怀孕晚期流产,流产犊牛全部为母犊。结果产下1公1母两头犊牛,流产个体与出生个体的性别与PCR鉴别结果完全相符。

节瓜ISSR-PCR反应体系的建立与正交优化

旨在开展节瓜种质资源分类鉴定与遗传多样性研究。通过正交试验设计与单因素分析相结合的方法,对节瓜ISSR-PCR反应体系5个因素(模板DNA浓度、dNTP浓度、Mg2+浓度、引物浓度与Taq聚合酶浓度)在4个水平上进行优化分析,建立了节瓜稳定可靠且具丰富多态性的最佳反应体系,进而对引物退火温度进行梯度试验分析。结果表明,20μL节瓜ISSRPCR最佳反应体系为70 ng模板DNA、0.2 mmol/L dNTP、1.2 mmol/L Mg2+浓度、0.96μmol/L引物、0.8 U Taq DNA聚合酶和2.0μL10×buffer;引物IS807最佳退火温度为53℃。以此为基础,利用4条引物对4份节瓜种质进行最佳反应体系稳定性验证,证明该体系稳定可靠、扩增谱带清晰、多态性丰富且重复性较好。

二温式RT-PCR检测对虾Taura综合征病毒的研究

设计了一对能扩增大小为231bp对虾Taura综合征病毒 (TSV)某段基因的特异性引物 ,优化建立了能快速检测TSV的二温式RT-PCR。特异性和敏感性试验结果表明 ,二温式RT-PCR能对3个试验用TSV毒株的RNA进行扩增 ,并得到与预期大小一致的231bp的扩增产物 ,而其它3种对虾病原则无相同大小的特异性扩增产物出现 ;RT-PCR最低能检测到1pg的TSVRNA。应用RT-PCR对320份分别来自广西沿海不同对虾养殖场的对虾样品进行检测 ,结果一共有85份检出TSV。这说明了TSV在广西沿海地区养殖对虾中已呈现出区域性流行趋势 ,同时也显示了RT-PCR在TSV临床检测中具有较高的实用性。

二温式PCR检测对虾白斑综合征病毒

本研究设计了一对能扩增大小为 30 6 bp对虾白斑综合征病毒 (WSSV)某段基因的特异性引物 ,优化建立了能快速检测 WSSV的二温式 PCR。在对包括 10 5份临床样品、10份 SPF南美白对虾组织样品和其他对虾病害病原在内的样品检测结果中 ,有 6 5份临床样品呈现 WSSV阳性 ,而 10份 SPF南美白对虾组织样品和其他对虾病害病原的 PCR结果为阴性。该二温式 PCR最低能检测到 1pg的 WSSV感染对虾组织样品总 DNA。这些结果表明 ,该 PCR具有高度的特异性和敏染性。

PCR芯片控温系统研究

通过采用以单片机为核心的数字电路控温技术、PWM技术以及PID控温算法 ,研制出成本低、控温精度高。

牛环形泰勒虫病二温式PCR诊断方法的建立及初步应用

为建立特异、敏感、快速的二温式PCR诊断方法,根据牛环形泰勒虫裂殖子表面抗原(tams1)基因,设计了一对特异性引物,扩增出大小为154bp基因片段,经克隆、测序分析,与已知基因序列的相似性为96%。用建立的牛环形泰勒虫病二温式PCR诊断方法,对从新疆牛环形泰勒虫病流行地区采集的50份全血样品进行诊断,阳性率为88%,而血涂片检出的阳性率只有58%。经试验验证,该方法具有特异性高、敏感性强、重复性和稳定性好等优点。表明本试验所建立的二温式PCR诊断方法可用于牛环形泰勒虫病的临床诊断、隐性感染检测和流行病学调查。

基因扩增分析(PCR)仪温控系统的研究与应用

分析了基因扩增分析(PCR)仪的工作原理及提高PCR仪温度控制精度的技术,详细讨论了温度控制系统的软硬件设计,研制了一种新颖的PCR温度控制系统。

5种温带果树SRAP-PCR退火温度的优化

SRAP技术是一种多态性和信息量丰富的新型分子标记技术,近年来在植物遗传多样性分析、种质鉴定、遗传连锁图的构建以及比较基因组学研究等方面得到广泛应用。有关SRAP-PCR反应体系的优化很少涉及到退火温度。因此,我们利用梯度PCR技术,对SRAP-PCR过程中的两步退火温度进行了研究,以期获得较好的退火温度组合。通过对第一步前5轮循环的退火温度梯度37～50℃的实验,其退火温度可提高到41℃;对第二步后30轮循环的退火温度梯度50～60℃的实验,其退火温度可提高到52℃。应用该程序在苹果、桃、板栗、梨和樱桃等5个树种各4个品种进行扩增,均获得了良好的结果。

牛支原体、牛病毒性腹泻病毒和牛传染性鼻气管炎病毒三重二温式PCR检测方法的建立及应用

为建立一种可同时检测牛支原体(MB)、牛病毒性腹泻病毒(BVDV)和牛传染性支气管炎病毒(IBRV)的三重二温式PCR检测方法。根据GenBank中MB、BVDV和IBRV的保守基因Uvrc基因、5′端非编码区(5′-UTR)和GB基因,分别设计了3对特异引物,将三温式PCR扩增程序简化为两个温度梯度,优化反应条件建立了用于同时检测MB、BVDV以及IBRV 3种常见牛呼吸道传染病病原的三重二温式PCR。结果显示,该方法特异性好,只对MB、BVDV和IBRV模板进行扩增,扩增的目的片段长度分别为412、170、727bp,对其他牛病原体无特异性扩增;灵敏度高,最低能同时检测到10 000拷贝的目的核酸;干扰性小,能同时检测3个不同浓度的模板。研究所建立的MB、BVDV和IBRV三重二温式PCR检测方法,具有特异性好、灵敏度高、方便快速等优点,可用于临床鉴别诊断和流行病学调查,具有很高的临床应用价值。

应用二温式PCR检测诊断爱德华氏菌病

根据爱德华氏菌(Edwardsiella)的16S rDNA基因序列设计一对特异性引物,用二温式PCR对6株爱德华氏菌均扩增出与预期大小相一致的576 bp产物,而对嗜水气单胞菌(Aeromonas hydrophila)、温和气单胞菌(Aeromonas sobria)、荧光假单胞菌(Pseudcmonas fluoroscercs)、柱状屈挠杆菌(Cytophaga columnaris)、链球菌(Streptococcus)、葡萄球菌(Staphylococci)、弧菌(Vibrio)、大肠杆菌(Escherichia colibacillus)和沙门氏菌(Salmonella)等10种病原体的扩增,结果全为阴性。该二温式PCR可以检测到1 pg的爱德华氏菌DNA模板和48个菌体。本实验建立的二温式PCR为爱德华氏菌病的早期诊断与有效的防治提供了快速检测方法,对水产品的食品安全有重要的意义。