Taqman MGB探针分型技术检测HBV YMDD变异及其临床应用

目的:建立一种快速准确的HBV YMDD变异检测方法,用于乙肝患者拉米夫定治疗中耐药性突变的监测.方法:采用三种特异性Taqman MGB探针和一对共同引物,分别针对总HBV、YIDD变异、YVDD变异的三管并行检测,以确定YMDD变异的类型及其在病毒群中的比例.以序列直接测定法作为诊断YMDD变异的金标准,对该方法YMDD变异检测的灵敏度、特异性和诊断符合率作评价.并对112例经拉米夫定治疗的血清HBV DNA持续阳性的乙型肝炎患者检测其YMDD变异情况.结果:该方法检测YIDD、YVDD的最低检测界限为1 000 Copies/mL,能在106 Copies/mL病毒群中检出0.1%的变异株的存在.以直接测序方法为金标准,该方法YMDD变异检测的灵敏度为100%、特异性为96%,诊断符合率为97.5%.该方法检测112例患者37例发生YMDD变异,阳性率为27.2%.结论:该方法能够直接检测HBV YMDD变异的类型及其在病毒群中的比例,具有快速、灵敏、准确、经济实用等优点,适合临床实验室开展拉米夫定耐药性突变的监测.     

双探针实时荧光定量PCR检测对拉米呋啶耐药的乙型肝炎患者HBV变异

目的研究双探针实时荧光定量PCR方法检测对拉米夫定耐药的乙肝患者血清中HBV病毒变异的可行性。方法用双探针MGB实时荧光定量PCR法检测拉米夫定治疗后的乙型肝炎患者血清,确定HBV YMDD变异类型,与测序法进行比对。结果68例标本中本法测出27例YMDD野生株,20例Y IDD变异株,11例YVDD变异株,与直接测序结果一致。另有10例标本测得为混合株,经克隆后测序证实,混合株中存在优势株。直接测序只能检测出优势株。结论双探针MGB实时荧光定量PCR法可以快速、准确地测出HBV DNA混合变异毒株。     

泉州地区拉米夫定抗乙肝病毒治疗YMDD变异

目的 检测泉州地区乙肝病毒YMDD变异的类型,探讨拉米夫定治疗后及未接受拉米夫定治疗的乙型肝炎患者中乙肝病毒(HBV)发生YMDD变异情况.方法 采用聚合酶链反应(PCR)结合Taqman荧光技术对泉州地区114例慢性乙肝患者进行HBV YMDD检测,一组66例接受拉米夫定治疗及保肝治疗,另一组48例为对照组只给予保肝治疗,治疗1年对患者HBV多聚酶基因YMDD基序定性检测,观察是否有突变.结果 拉米夫定治疗组在用药1年后有24例发生YMDD突变.其中YVDD变异有15例,YIDD变异有6例,YIDD YVDD变异有3例,其中变异株中有15例,伴有HBV DNA、ALT高于正常水平;而对照组有17例发生YMDD突变.其中YVDD变异有9例,YIDD变异有4例,YIDD YVDD变异有4例.结论 泉州地区乙肝病毒变异株主要是YVDD型,在未经抗病毒治疗的慢性乙肝患者中存在YMDD变异株,临床上在应用拉米夫定对慢性乙型肝炎惠者进行抗病毒治疗前,应对患者是否存在YMDD变异加以重视.     

G6PD缺乏症基因诊断方法的建立

目的：建立有效可行的葡萄糖‐6‐磷酸脱氢酶（glucose‐6‐phosphate dehydrogenase ,G6PD）缺乏症基因突变型检测的经济、快速、无污染的实用方法。方法建立多重 Taqman MGB 探针荧光 PCR体系,对6种国内主要基因突变型（1376G＞ T、1388G＞ A、95A＞G、871G＞A、392G＞ T、1024C＞ T ）进行突变检测,同时加入βactin基因作内参对整个PCR体系进行监控。用30份已知基因型样品进行重复性检测实验。对200例未知样本同时用多重 Taqman MGB探针荧光法和DNA直接测序法进行检测验证。结果建立的多重Taqman MGB探针荧光PCR法同时检测6个国内最常见G6PD基因突变位点,可在1小时内出检测结果,批内与批间的重复性均为100％。200例未知基因型样本检出34例突变,Taqman MGB探针荧光 PCR法分型结果与DNA直接测序法检测结果一致,阳性符合率、阴性符合率均为100％。结论建立的多重Taqman MGB探针荧光PCR法,可在1小时内出结果,PCR后无需开管,是一种快速、有效可行的G6PD缺乏症基因诊断方法。     

海南地区拉米夫定抗乙肝病毒治疗YMDD变异

目的：探讨拉米夫定治疗后乙肝病毒发生YMDD变异情况，及未接受拉米夫定治疗的乙型肝炎患者中是否存在天然变异的情况。方法：采用聚合酶链式反应（PCR）结合Taqman荧光技术对海南地区110例慢性乙肝患者进行HBV YMDD检测，一组80例接受拉米夫定治疗及保肝治疗，另一组30例为对照组只给予保肝治疗，治疗1年对患者HBV多聚酶基因YMDD基序定性检测，观察是否有突变。结果：拉米夫定治疗组在用药1年后有31例发生YMDD突变．其中YIDD变异有15例，YVDD变异有7例，YIDD＋YVDD变异有9例，变异株中有19例伴HEV DNA、ALT水平高于正常水平；而对照组有11例发生YMDD突变．其中YIDD变异有2例．YVDD变异有1例．YIDD＋YVDD变异有8例。结论：在未经抗病毒治疗的慢性乙肝患者中存在YMDD变异株，临床上在应用拉米夫定对慢性乙型肝炎患者进行抗病毒治疗前．应对患者是否存在YMDD变异加以重视。     

拉米夫定抗乙肝病毒治疗YMDD变异研究

目的]探讨拉米夫定治疗后乙肝病毒发生YMDD变异情况，及未接受拉米夫定治疗的乙型肝炎患者中是否存在天然变异的情况。[方法]采用聚合酶链式反应（PCR）结合Taqman荧光技术对海南地区110例慢性乙肝患者进行HBVYMDD检测，一组80例接受拉米夫定治疗及保肝治疗，另一组30例为对照组只给予保肝治疗，治疗1年对患者HBV多聚酶基因YMDD基序定性检测，观察是否有突变。[结果]拉米夫定治疗组在用药1年后有31例发生YMDD突变，其中YIDD变异有15例，YVDD变异有7例，YIDD＋YVDD变异有9例，变异株中有19例伴HBVDNA、ALT水平高于正常水平；而对照组有11例发生YMDD突变，其中YIDD变异有2例，YVDD变异有1例，YIDD＋YVDD变异有8例。[结论]在未经抗病毒治疗的慢性乙肝患者中存在YMDD变异株，l临床上在应用拉米夫定对慢性乙型肝炎患者进行抗病毒治疗前，应对患者是否存在YMDD变异加以重视。     

Taqman MGB探针分型技术检测HBV YMDD 变异及临床应用研究

摘　要: 目的：建立一种快速准确的HBV YMDD变异检测方法，用于乙肝患者拉米夫定治疗中耐药性突变的监测。方法：采用三种特异性TaqmanMGB探针和一对共同引物，建立了检测HBV YMDD变异的荧光定量PCR方法。分别针对总HBV 、YIDD变异、YVDD变异的三管并行检测，可以确定YMDD变异的类型及其在病毒群中的比例。对YMDD野生株、YIDD变异株、YVDD变异株的病毒质粒经过梯度稀释进行检测验证。以序列直接测定法作为诊断YMDD 变异的金标准，对该方法YMDD变异检测的灵敏度、特异性和诊断符合率作评价。并对112例经拉米夫定治疗的血清HBV DNA持续阳性的乙型肝炎患者检测其YMDD变异情况。结果　该方法在105--108Copies/ml 的YMDD野生株、YIDD变异株、YVDD变异株检测中匀未出现非特异性扩增信号；相同浓度的YMDD、YIDD、YVDD在三种不同探针的反应管中的扩增效率基本一致；病毒野生株与YVDD变异株的实际混合比例与计算比例基本一致；该方法检测YIDD、YVDD的最低检测界限为1000 Copies/ml，能在106Copies/ml病毒群中检出0.1%的变异株的存在。以直接测序方法为金标准，该方法YMDD变异检测的灵敏度100%、特异性96%，诊断符合率97.5%。该方法检测112例经拉米夫定1--3年治疗血清HBV-DNA持续阳性的乙型肝炎患者37 例发生YMDD 变异,阳性率27.2%。结论：该方法能够直接检测HBV YMDD变异的类型及其在病毒群中的比例，具有快速、灵敏、准确、经济实用等优点，适合临床实验室开展拉米夫定耐药性突变的监测。     

实时荧光PCR监测HBV感染患者拉米夫定治疗病毒YMDD变异

目的 建立一种快速准确的实时荧光PCR方法检测HBV感染患者经拉米夫定治疗后YMDD变异情况.方法 首先用PCR法筛选HBV DNA阳性血清157例,HBV DNA含量为2.0×103～8.0×108拷贝/ml,HBV DNA阴性血清30例,然后采用TaqMan探针技术和选择性引物的实时荧光PCR对其进行YMDD变异检测.通过变异株和对照管的循环阈值(cycle threshold,Ct值)差来计算血清中总HBV中的YMDD变异株含量,并用PCR产物直接测序方法随机对69例HBV DNA阳性标本RT区进行基因序列分析,验证所建立方法的准确性.结果 187例接受拉米夫定治疗患者血清标本中,发生YMDD变异88例,其中YIDD变异39例,YVDD变异38例,YIDD与YVDD混合变异11例.突变株的含量为10%～100%.30例HBV DNA阴性血清的C管循环40次,69例测序结果显示68例两种方法检测结果完全相同,符合率为98.55%.结论 采用选择性引物和TaqMan探针技术建立实时荧光PCR方法能快速准确地检测YMDD变异情况,并能检测患者变异株在HBV总量中的比例,为临床使用拉米夫定治疗乙型肝炎病毒感染监测耐药性提供一种有效方法.     

拉米夫定治疗慢性乙型肝炎患者乙型肝炎病毒YMDD变异的研究

目的探讨拉米夫定治疗1年后慢性乙型肝炎（CHB）患者发生YMDD基序变异的状况及其检测方法。方法采用荧光定量PCR法检测血清HBV DNA含量，荧光PCR法和变性高效液相色谱法检测HBV YMDD基序变异。结果61例CHB患者经过拉米夫定治疗1年后，40例（65．6％）HBV DNA阴转；在21例HBV DNA阳性患者中，10例为YMDD野生型，11例出现YMDD基序变异，变异率达18．0％（11／61）。在11例基序变异中，完全变异7例（63．6％），部分变异4例（36．4％）。在完全变异型中，YIDD变异型5例，YVDD变异型2例；在部分变异型中，YIDD变异型3例，YVDD变异型1例。结论CHB患者拉米夫定治疗1年后，出现较高的YMDD基序变异率。利用荧光PCR法结合变性高效液相色谱法检测基序变异，经济便捷，可作为CHB患者YMDD基序变异的常规监测。     

1000例慢性乙型肝炎应用拉米夫定治疗YMDD变异的综合分析

目的 研究分析慢性乙型肝炎在长期应用拉米夫定治疗中,HBV YMDD发生变异的影响因素.方法 采用聚合酶链式反应(PCR)结合Taqman荧光探针技术,对1000例慢性乙型肝炎血清中乙肝病毒(HBV DNA)多聚酶YMDD进行检测,并分析患者用药时间、用药前HBV DNA载量、ALT水平、患者的性别和年龄等与之相关性.结果 1000例慢性乙型肝炎服用拉米夫定半年以上的患者检出YMDD变异134例913.4%);其中.YMDD无变异者治疗前ALT水平显著高于变异者(P＜0.05);而YMDD变异者治疗前HBV DNA含量显著高于无变异者(P＜0.05),男性组(13.0%)与女性组(15.2%)的变异率差异无显著性(P＞0.05);HBeAg阳性组变异率(13.9%)与HBeAg阴性组(13.4%)相比差异无显著性(P＞0.05);不同年龄组之间差异无显著性(P＞0.05).结论 HBV多聚酶基因YMDD变异可能与患者治疗前HBV DNA含量、ALT水平和用药时间有关系,而与患者的性别、年龄和血清HBeAg状态无关.     

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.