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.

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。

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.

一种分区温控的实时荧光PCR快速热循环系统设计

热循环系统是实时荧光聚合酶链式反应(PCR)仪的关键组成部分,决定了核酸检测效率和结果准确性.针对传统PCR检测系统的热循环耗费时间长、温度控制复杂的问题,设计了一种分区温控的实时荧光PCR热循环系统,包括热循环系统硬件电路和机械结构,通过控制试液在不同恒温区切换实现快速热循环,采用增量式比例积分微分算法控制恒温区温度,控制精度达到±0.1℃.使用Fluent建立传热模型,分析试液热延迟现象预估试液变化规律.通过搭建PCR样机进行实验验证,试液升降温速率分别为3.8和4.4℃/s,证明了所提出PCR热循环系统能有效提高检测效率.

基于物联网的PCR温度控制系统设计

核酸检测在疫情防控中发挥着重要作用,目前商品化PCR仪通常需要专业的技术人员和实验室,耗费大量的时间和人力。本文设计了基于物联网的PCR温度控制系统,能够实现快速、准确的温度控制。通过将检测设备端接入机智云平台,使检测人员能够远程监控PCR仪的运行,实现检测现场的无人值守,减轻检测人员的负担,并提高检测的效率。

BVDV与IBRV二重二温式PCR检测方法的建立及应用

针对牛病毒性腹泻病毒(BVDV)的5′非翻译区(5′UTR)和牛传染性鼻气管炎病毒(IBRV)的gB基因分别选取2对引物,建立这2种病毒的二重二温式PCR检测方法,该方法特异性高,可检出BVDV与IBRV,与牛轮状病毒等均无交叉反应;对BVDV与IBRV的最低检测质量浓度为1.32、0.047 mg/L。使用该方法对采集自宁夏地区的113份具有BVDV与IBRV临床症状的牛的粪便进行检测,结果显示BVDV与IBRV的阳性检出率分别为12.39%、22.12%,2种病原体混合感染的阳性检出率为7.08%。与单重二温式PCR检测结果进行比较,得出BVDV阳性符合率为93.33%,IBRV阳性符合率为92.53%,2种病原体混合感染的阳性符合率为100%。

非洲猪瘟病毒微滴式数字PCR检测用引物和探针的筛选

本试验针对非洲猪瘟病毒基因保守序列设计了四对引物和探针用于微滴式数字PCR检测,经过数字PCR梯度反应体系及条件优化、特异性试验、灵敏度和线性范围试验,最终确认了一对引物和探针结果较为理想,其中引物浓度为900 nmol/L,探针浓度450 nmol/L,最适退火温度为54.2℃。

荧光定量PCR仪温度控制系统

介绍了两种常见的用于荧光定量PCR仪的温度控制系统。首先概述了采用经典的比例-积分-微分控制算法的温度控制系统,结合了自动调谐算法,具有易实现和循环时间短等优点,但是难以适应荧光定量PCR仪器高准确度的要求。因此,又介绍了一种荧光定量PCR仪器的多模态温度控制系统,采用了模糊PID控制算法,与第一种温度控制系统相比,该系统具有较短的稳态时间、较小的超调量、较强的鲁棒性和较高的准确度。介绍的两种温度控制系统对进一步开发利用荧光定量PCR仪的测试校准工作具有一定的参考价值。

基于PID算法的PCR仪温度控制系统研制

聚合酶链式反应需高温变性、低温退火、适温延伸3个阶段,所以对PCR仪温度的精确控制是最主要问题。提出一种运用PID算法控制PCR温度,由MCU单片机产生定周期可变占空比信号经功率放大电路,驱动半导体制冷片进行仪器的升降温,增加热盖电路防试剂蒸发、增加温度补偿电路补偿PCR温度。运用专用测温工具测量PCR仪器温度,实验结果表明:所设计的温度控制系统升降温的平均速率可达3℃/s,控温精度可达±0.2℃内,控温稳定,具有较好的应用效果。

PCR检测仪温度控制系统的设计与仿真

近年来,随着病毒的频繁发生和检测需求的急剧增加,PCR检测仪越来越受到关注。鉴于目前PCR检测仪的温度控制系统温控速率和温控精度不够的现象。本文以STM32为主控制芯片,搭建了温度控制电路,并在传统PID控制器的基础上引入模糊控制器组成模糊PID控制器,弥补传统PID算法参数整定困难、响应速度慢等缺点。在SIMULINK环境下建立了仿真模型,仿真结果表明模糊PID控制器的性能优于传统PID控制器。

qPCR仪T_(m)校准的影响因素及结果分析

qPCR仪熔解曲线校准在物理参数层面,可更加直观地反应荧光定量PCR仪温控及光学系统的可靠性及相互影响,是全面评估荧光定量PCR仪的校准手段。由于熔解过程中,预熔解至熔解阶段的温度爬升速率不同,导致校准结果差异,因此,本文通过实验设计,分析了不同熔解爬升速率条件下Tm校准结果的差异和熔解曲线校准法的注意事项,为qPCR仪综合物理参数校准提供了参考。

PCR仪温度场无线校准装置的研制及性能验证

目的:为满足日益增长的PCR仪温度场校准需求,解决校准装置被国外技术垄断且适用范围有限等问题,研制温度场无线校准装置。方法:采用低功耗蓝牙技术,以嵌入式系统为核心,实现16路高精度温度数据采集、无线传输及分析处理,并依据JJF1821-2020《聚合酶链反应分析仪温度校准装置校准规范》对PCR仪温度场无线校准装置进行性能验证。结果:经校准溯源,该装置温度测量不确定度U=0.06℃(k=2),满足相关校准规范要求。结论:无线校准装置满足96孔PCR仪温度场校准要求,实现了技术完全自主可控。

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。