微孔板杂交法检测结核分枝杆菌的初步临床应用

目的:对PCR-微孔板杂交法的临床应用进行评价.方法:取临床标本268份分别进行抗酸染色、培养、PCR扩增产物电泳、PCR微孔板杂交四种方法的比较.结果:在216份临床高度怀疑有结核分枝杆菌感染的患者标本中,PCR-微孔板杂交法检出阳性为98例,其检出率高于PCR-电泳法(91/218)、培养法(81/218)和抗酸染色法(56/218).50份临床证实无结核分枝杆菌感染的病人标本,四种方法均未能检出结核分枝杆菌.结论:PCR-微孔板杂交法是高度敏感、高度特异的快速检测结核分枝杆菌方法.     

PCR-微孔板反向杂交法在结核分枝杆菌检测中的应用

目的评价PCR-微孔板反向杂交法检测临床标本中结核分枝杆菌(MTB)的应用价值.方法应用集菌涂片镜检法、培养法、PCR法、PCR-微孔板反向杂交法分别对55份肺结核病患者和30份非结核呼吸系统病患者痰标本平行检测.结果涂片法对结核病患者临床标本中MTB检出率为21.82%,培养法检出率为25.46%,PCR法检出率36.36%,PCR-微孔板反向杂交法检出率47.27%.对30例非结核呼吸系统疾病患者检测MTB,PCR法出现2例假阳性,其余3种方法均阴性,PCR-微孔板反向杂交法比单纯PCR法灵敏度高、特异性强.结论PCR-微孔板反向杂交法具有简便、快速、抗污染、灵敏度高、特异性强的优点,缺点是存在假阴性,应改进标本前处理方法,在对结核病的辅助诊断中应与细菌学方法相结合,以提高检出率.     

多重聚合酶链反应微孔板杂交法在性病病原体检测中的应用

目的探讨敏感、特异的同时能检测3种性病病原体的方法。方法采用3种不同的特异性引物与梅毒螺旋体(TP)、单纯疱疹病毒(HSV)和杜克雷杆菌(HD)DNA模板进行单一和多重聚合酶链反应(M-PCR)、微孔板杂交,用不同梯度的DNA模板及其它常见16种泌尿生殖道微生物标本验证其敏感性和特异性。结果3种不同的特异性引物在单一和M-PCR反应中均能特异地扩增,其扩增产物均能与相对应的特异探针杂交;单一和M-PCR微孔板杂交法均能检测出单个和3种病原体靶基因10个拷贝,而其它常见泌尿生殖道病原微生物交结果均为阴性。结论M-PCR微孔板杂交法能同时敏感、特异地检测TP、HSV和TP性病病原体。     

PCR-微板杂交-ELISA检测结核杆菌DNA

[目的]探讨PCR-微板杂交-ELISA方法检测结核杆菌DNA的临床价值。[方法]选择PCR扩增区域内的保守序列设计捕获探针和显色探针,分别用于DNA杂交和ELISA分析,建立PCR-微板杂交-ELISA方法并运用该方法对328份结核病人痰标本进行了检测。[结果]328份结核病人痰标本中有196例结核杆菌DNA阳性,阳性率为59．7％。检出率明显高于痰培养和痰涂片抗酸染色。[结论]PCR-DNA微板杂交-ELISA方法综合运用了PCR、DNA杂交和-ELISA技术,大大增加了DNA检测的特异性和灵敏度,在临床上具有良好的应用前景。     

HLA分型基因微阵列的构建及与PCR-SSO、PCR-SSP、SBT的比对

目的建立HLA分型基因微阵列新技术，并与PCR-SSO（PCR-顺序特异性寡核苷酸）、PCR-SSP（PCR-序列特异性引物）及SBT（DNA测序）进行方法学比对。方法基于反向SSO原理设计引物和探针→探针点样→标本处理和标记→杂交检测分析，其依据的原理和主要步骤为：（1）PCR扩增；（2）DNA杂交；（3）酶联染色；（4）结果判断。以此构建HLA-B27分型基因微阵列系统，并检测88例标本，将此结果与PCR-SSO、PCR-SSP及金标准的SBT三种方法进行比对。结果成功构建了HLA-B27分型基因微阵列系统。其与PCR-SSO、PCR-SSP及SBT方法相比总体一致率为98．86％；B27阳性标本结果的一致率为97．72％（86／88）；B27阴性标本结果的一致率为100％（4／4）。结论与目前常用的PCR-SSO，PCR-SSP及SBT等方法相比，HLA分型基因微阵列具有高效、快速、稳定、经济等特点，可同时进行HLA-B27基因检定与等位基因分型，不失为临床及造血干细胞库大规模HLA分型（中低分辨）的一种新方法。     

多重PCR快速鉴定19种致病真菌

摘要:目的：建立快速鉴定致病真菌的多重PCR体系。 方法：选取19种致病真菌的rDNA内部转录间隔区（ITS）序列设计引物,建立两个多重PCR体系,扩增临床与环境分离真菌菌株、人类细胞及9种常见细菌DNA,检测体系的特异性;以浓度梯度的DNA模板检测体系的灵敏度。 结果：对氟康唑天然耐药的克柔念珠菌、光滑念珠菌以及对两性霉素B耐药的土曲霉、镰刀菌、尖端赛多孢、根霉在该体系中可扩增出特异性条带,其他真菌可同时进行种属鉴定。74株被测真菌菌株的鉴定结果与常规鉴定的符合率为95.9%。两个多重PCR体系对人类基因组及9种常见细菌DNA的扩增结果均为阴性;扩增阳性的最低模板浓度均为10 fg/μL。 结论：建立的两个多重PCR体系可对致病真菌进行检测和种属鉴定。     

人乳头瘤病毒PCR产物酶标-微孔板杂交分型

目的建立聚合酶链反应（ＰＣＲ）产物直接酶标记微孔板反向分子杂交法用于人乳头瘤病毒（ＨＰＶ）型别鉴定。方法利用ＨＰＶ通用引物介导ＰＣＲ（ＧＰＰＣＲ）扩增靶ＤＮＡ，然后对产物直接标记辣根过氧化物酶复合物（ＨＲＰＰＥＩＱＩ），与预先包被在微孔板上的ＨＰＶ寡核苷酸探针杂交后酶显色法检测。结果ＨＰＶ各型之间无交叉杂交；杂交灵敏度可达１３～７６个病毒ＤＮＡ拷贝，比ＰＣＲ琼脂糖凝胶电泳检测高１０倍；该法重复性好，阳性孔批内ＣＶ为６．３４％，批间ＣＶ为１０．５３％，阴性孔批内ＣＶ为１％，批间ＣＶ为５．７９％；而且杂交影响因素较少。结论该法特异性强、灵敏度高、操作简便，无放射性和ＥＢ染料污染，杂交时间短、仪器读取结果，便于大量常规临床样本定性或定量检测。     

微孔板杂交法检测结核分枝杆菌的初步临床应用

目的：对ＰＣＲ－微孔板杂交法的临床应用进行评价。方法：取临床标本２６８例分别进行抗酸染色、培养、ＰＣＲ扩增产物电泳、ＰＣＲ微孔板杂交四种方法的比较。结果：在２１６份临床高度怀疑有结核分枝杆菌感染的患者标本中,ＰＣＲ－微孔板杂交法检测阳阳性为９８例,其检出率高于ＰＣＲ－电泳法（９１／２１８）、培养法（８１／２１８）和抗酸染色法（５６／２１８）,５０份临床证实无结核分枝杆菌感染的病人标本,四种方法均未     

一管式多引物焦磷酸测序技术在HPV分型检测中的应用

目的：探讨一管式多引物焦磷酸测序技术在人类乳头瘤病毒（HPV ）分型检测中的临床应用价值。方法设计针对7个不同 HPV亚型的高度特异性测序引物,采用一管式多引物测序法,通过半巢式聚合酶链反应（PCR）扩增获得 HPV基因片段,并进行焦磷酸测序分析。结果通过半巢式PCR扩增和一管式多引物焦磷酸测序,成功区分了7种不同的 HPV亚型,且测序结果与PCR-反向点杂交法检测结果的符合率是100％。结论一管式多引物焦磷酸测序技术是一种简便、高效、高通量、高灵敏度、高准确度的HPV分型检测方法,值得推广应用。     

改进的多重PCR技术鉴定白色念珠菌、新生隐球菌及烟曲霉的实验研究

[目的]建立一种快速而简便的鉴定白色念珠菌、新生隐球菌及烟曲霉的分子生物学方法。[方法]应用通用引物ITS1与ITS4，PCR扩增真菌的内转录间隔区（包含5．8SrDNA），然后将白色念珠菌、新生隐球菌、烟曲霉的单条种特异性异物与引物ITS1结合，对通用引物的扩增产物进行多重PCR扩增，达到鉴定上述菌株的目的。[结果]实验所采用的所有致病真菌，经通用引物的PCR扩增均能扩增出特异性的条带，而非真菌菌株扩增为阴性；白色念珠菌、新生隐球菌、烟曲霉的3种种特异性引物与ITS1结合进行的多重PCR扩增，只对目的菌株进行了有效扩增，其他菌株扩增为阴性。应用此方法对临床分离的菌株，白色念珠菌，新生隐球菌，烟曲霉进行鉴定，也得到特异性扩增条带。[结论]改进的多重PCR技术，更加经济，简单，快速，为致病真菌菌种的鉴定提供了有效手段。     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Dissociable correlates of two classes of retrieval processing in prefrontal cortex

Although substantial evidence suggests that the prefrontal cortex (PFC) implements processes that are critical for accurate episodic memory judgments, the specific roles of different PFC subregions remain unclear. Here, we used event-related functional magnetic resonance imaging to distinguish between prefrontal activity related to operations that (1) influence processing of retrieval cues based on current task demands, or (2) are involved in monitoring the outputs of retrieval. Fourteen participants studied auditory words spoken by a male or female speaker and completed memory tests in which the stimuli were unstudied foil words and studied words spoken by either the same speaker at study, or the alternate speaker. On "general" test trials, participants were to determine whether each word was studied, regardless of the voice of the speaker, whereas on "specific" test trials, participants were to additionally distinguish between studied words that were spoken in the same voice or a different voice at study. Thus, on specific test trials, participants were explicitly required to attend to voice information in order to evaluate each test item. Anterior (right BA 10), dorsolateral prefrontal (right BA 46), and inferior frontal (bilateral BA 47/12) regions were more active during specific than during general trials. Activation in anterior and dorsolateral PFC was enhanced during specific test trials even in response to unstudied items, suggesting that activation in these regions was related to the differential processing of retrieval cues in the two tasks. In contrast, differences between specific and general test trials in inferior frontal regions (bilateral BA 47/12) were seen only for studied items, suggesting a role for these regions in post-retrieval monitoring processes. Results from this study are consistent with the idea that different PFC subregions implement distinct, but complementary processes that collectively support accurate episodic memory judgments.     

俯卧位通气对高海拔地区肺复张治疗无效急性呼吸窘迫综合征患者氧合的影响

目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高＜20％视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P＜0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P＜0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P＜0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.     

Digital imaging among medical facilities

The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.     

Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts

Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.     

Clinical Pharmacokinetics of Morphine

Morphine, the most widely used mu-opioid analgesic for acute and chronic pain, is the standard against which new analgesics are measured. A thorough understanding of the pharmacokinetics of morphine is required in order to safely and effectively use this analgesic in a wide variety of patients with different levels of organ function. A MEDLINE search was conducted to identify literature published between 1966 and January 2002 relevant to the pharmacokinetics of morphine. These publications were reviewed and the literature summarized regarding unique and clinically important elements of morphine disposition relative to its parenteral administration (including intravenous, intramuscular, subcutaneous, epidural and intrathecal administration), absorption profile (immediate release, controlled release, and sublingual/buccal, and rectal administration), distribution, and its metabolism/ excretion. Special populations, including infants, elderly, and those with renal/liver failure, have a unique morphine pharmacokinetic profile that must be taken into account in order to maximize analgesic efficacy and reduce the risk of adverse events.