基于微流控核酸等温扩增的 登革病毒现场快速检测技术研究

[摘要]?目的?结合环介导等温扩增技术（loop-mediated isothermal amplification, LAMP）和微流控芯片技术，建立一种适合现场快速检测登革病毒的方法。方法?利用RT-LAMP，针对登革病毒基因组中3’非编码区中特异性序列进行扩增，建立基于微流控芯片技术的LAMP检测方法，优化检测体系，并对该方法的灵敏性和特异性进行评估。结果?基于LAMP 和微流控芯片技术的登革病毒检测方法，通过对病毒模板进行扩增，发现与其他病毒无交叉反应，特异性良好；同时，结果显示该方法对登革病毒检测灵敏性可达61.2 pg/μl，与实时荧光定量PCR仪所达到的检测灵敏性一致。结论?基于LAMP和微流控芯片技术的登革病毒检测方法具有操作简单、快速、对设备要求低等优势,并且灵敏性、特异性均较好，是一种便于开展现场快速检测的方法。     

双重实时荧光PCR快速鉴别人参和西洋参＊

目的 建立一种能够快速鉴定人参和西洋参的双重实时荧光PCR方法。方法 通过对人参属及其近似种基因序列的分析比对，设计特异性的引物探针，建立双重实时荧光PCR检测方法。PCR反应体系为Premix Ex Taq （Probe qPCR） 10 μL，人参上下游引物各0.5 μL，西洋参上下游引物各0.3 μL，人参与西洋参特异性探针各0.4 μL，DNA模板2 μL，灭菌去离子水补至20 μL。反应条件为95℃预变性30 s；然后以95℃变性5 s，60℃退火延伸30 s进行40个循环。应用该方法测试了27份DNA样品，包括7份人参、6份西洋参、4份人参与西洋参混合样、10份其他人参属样品及其他常见中药材样品的DNA，以确定该方法的特异性。将人参与西洋参样品DNA混合后10倍稀释成不同浓度后进行检测，用以确定该方法的灵敏度。结果 人参及西洋参样品均在特定的荧光通道下出现典型的阳性扩增曲线，人参与西洋参混合样品均同时出现两条阳性扩增曲线，其它对照样品及空白对照均没有出现阳性扩增曲线。灵敏度检测结果显示该方法检测人参及西洋参的最低检测限均为2 pg DNA/反应。结论 本实验建立的双重实时荧光PCR方法能够同时快速、准确、灵敏地鉴别出人参和西洋参。     

Utility of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for the rapid detection of nosocomial outbreaks of multidrug resistant organisms: Experience at a tertiary care center in North India

Background: There is a huge need to develop molecular typing methods which are simple to perform, rapid and cost effective to confirm clonality of nosocomial isolates in outbreak situations. Objectives: The aim of the study was to investigate a hospital outbreak of multi-drug resistant (MDR) Klebsiellapneumoniae septicemia in a paediatric surgery intensive care unit (PSICU) using a repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Materials and Methods: MDR Klebsiella pneumoniae isolates from an outbreak of nosocomial sepsis were typed byREP-PCR using consensus primers. Isolates from different intensive care units (ICUs) but with similar antibiogram were also genotyped for comparison. Results and Conclusion: A cluster of twelve MDR K Pneumoniae septicemia cases was identified at the PSICU by genotyping using REP-PCR. Surveillance cultures failed to pick up any source of infection. REP-PCR was found to be a rapid and simple tool for investigation outbreaks in hospitals. Due to early detection we could initiate infection control practices with focus on hand washing and prevent the further transmission of the organism.     

Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

个体化假体复合组织工程技术修复兔下颌骨缺损

目的研究快速成型（RP）技术辅助下制作的个体化假体复合珊瑚羟基磷灰石（CHA）、重组人骨形成蛋白2（rhBMP-2）修复兔下颌骨缺损的成骨效果。 方法以27只新西兰大白兔为实验对象，随机数字表法平均分成3组（每组9只），全部建立下颌骨连续性缺损模型，并在兔下颌骨缺损区分别植入个体化假体+自体骨（A组）、个体化假体+CHA（B组）、个体化假体+CHA+rhBMP-2（C组）。分别于术后4、12、24周3个时间点处死动物取材，进行大体标本观察，以及骨钙素（OC）、Ⅰ型胶原（COL-1）的免疫组化观察，分别比较各组修复骨缺损的能力，并对实验数据进行重复测量设计资料的单因素方差分析。 结果术后24周各组实验兔外形均对称，通过OC及COL-1的吸光度检测，骨缺损区均有大量新骨形成，A组（0.537 ± 0.010）、C组（0.530 ± 0.010）可见大量骨小梁及编织骨结构，缺损区的新骨OC、COL-1的免疫组化观察基本一致，差异无统计学意义（t = 0.007，P>0.05）；但A组强于B组（0.415 ± 0.009，t = 0.122，P<0.001）；C组也强于B组（t = 0.121，P<0.001），差异均有统计学意义。 结论在兔下颌骨缺损修复中，通过RP技术和组织工程技术相结合，CHA复合rhBMP-2后成骨能力明显增强，成骨效能肯定，为后期的临床应用提供可靠的实验基础。     

Recording of electroencephalograms and electrocardiograms during daytime sleep in trained canines: Preparation of the sleeping dogs

Abstract  Although respiration in trained canines is well investigated, the process of preparing dogs has not been described in any great detail. Moreover, their daytime patterns of sleep and wakefulness during 1 or 2 h of electroencephalogram (EEG) and electrocardiogram (ECG) recordings are not clear. Therefore, we describe the process of selecting and training dogs, in which we recorded EEG and ECG in the laboratory. First, 14 of 1242 dogs dealt with over a 1 year period were chosen. They were trained for 2 h to lie quietly and to sleep in the laboratory; this training procedure was repeated 152 times. Three dogs were then selected and a permanent tracheostomy was performed in one. Finally, EEG and ECG were recorded with the bipolar fine needle electrodes; respiration was recorded simultaneously through a tube inserted to a tracheostomy in one dog. Wakefulness, slow wave sleep (SWS) and rapid eye movement (REM) sleep (REMS) were identified according to the EEG pattern and on the basis of the behavioral criteria. Recordings were performed 12 or 13 times in each dog. Complete sleep cycles, including wakefulness, SWS and REMS in this sequence, were observed 3.9–4.1 times. The mean duration of SWS was 2.2–4.4 min and that of REMS was 3.5–4.6 min. The REMS latency was 33.9–41.8 min. Fluctuation of heart rate with respiration, termed respiratory sinus arrhythmia, was noted in the ECG. Heart beat increased with inspiration and decreased with expiration. The present study demonstrates how to select and train sleeping dogs and shows their undisturbed daytime sleep and wakefulness patterns.     

Spectral characteristics of early receptor potential in congenital red-green color blindness

The spectral response curve (amplitude versus wavelength) of the R₂ of the early receptor potential (ERP) was studied in normal, protan, and deutan subjects. The R₂ amplitude peaked at 520nm in most normal subjects. The R₂ at long wavelengths was smaller than normal in protans and larger than normal in deutans when the maximum amplitudes were normalized to 100% at the peak. The ratio of the R₂ amplitude at 460 nm to that at 600 nm clearly differed between protans and deutans. The ERP and the rapid off-response, which is mainly due to the cessation of the late receptor potential, were recorded in the same subjects. The ratio of the sensitivity of the rapid off-response at 500 nm to that at 600 nm was correlated with the ratio of the R₂ amplitude at 460 nm to that at 600nm (correlation coefficient, 0.823, p < 0.001). This study, in conjunction with our previous study, indicates that the abnormality is in the outer segments of the cones in protans and deutans.     

快速心房起搏对家兔L-型钙通道和Kv4.3钾通道基因表达的影响

目的观察快速心房起搏对家兔心房L型钙通道亚单位和Kv4.3钾通道基因表达的影响。方法新西兰大耳白家兔36只,随机分成6组,经右颈外静脉穿刺置入电极于右房,分别给予0、3、6、12、24或48h快速心房起搏,停止起搏后取右房组织,应用半定量反转录聚合酶链式反应测定各时相点L型钙通道α1c,β1,α2亚单位,钾通道Kv4.3mRNA的表达水平。结果L型钙通道α1c、β1亚单位在快速起搏6h后表达水平下调,并随着起搏时间的延长进一步下调。α2亚单位的mRNA表达在各时相点无显著差异。Kv4.3的mRNA的表达在快速起搏的24h和48h下降分别达55.50%（P<0.01）和59.12%（P<0.01）。48h后下降达到一个平台期。结论快速心房起搏可导致L型钙通道亚单位和Kv4.3钾通道mRNA表达下调。     

HIV抗体快速检测试剂的临床评估

目的:通过对HIV抗体快速检测试剂的临床评估,为HIV检测试剂的临床应用提供参考.方法:对快速试剂的使用性能进行比较,并采用美国输血协会(AABB)血清盘、临床样品血清盘、特性血清盘(阳性样品盘)、稀释系列血清盘评估快速试剂的敏感性和特异性.结果:三种快速试剂在检测AABB血清盘时,敏感性分别是86.4%、86.4%、100%,特异性皆为100%;三种快速试剂在检测临床样品血清盘时,敏感性分别是88.6%、91.4%、97.1%,特异性皆为100%.快速试剂具有很高的阳性预期值,对于低危人群(感染率很低人群)也具有很高的阴性预期值.快速试剂检测弱阳性的样品(酶联试剂s/co比值小于6～8的样品)存在漏检.快速试剂与ELISA参考试剂在分析灵敏度方面相差3个以上倍比稀释度.结论:快速试剂具有较好的使用特性,非常适用于样本量较少的实验室及对低危人群的HIV筛查,在对高危人群筛查时,可能有弱阳性样品漏检,同时快速试剂在分析灵敏度方面有待提高.