核酸薄膜技术快速检测结核分枝杆菌katG突变的研究

目的研制一种新的核苷酸薄膜,通过导流杂交技术检测结核分枝杆菌katG基因突变类型,以快速判断结核分枝杆菌对异烟肼的耐受性,寻找一种快速简便检测耐异烟肼结核病的方法。方法根据结核分枝杆菌katG基因序列设计1个野生型及2个常见突变型寡核苷酸特异探针,探针5’末端连接亚甲基(-CH2)。同时设计1个人类基因组探针以及1个生物素探针。制作低密度核苷酸薄膜微阵距列,通过导流杂交技术进行杂交,将杂交结果与DNA测序法对照。结果核酸测序显示,突变位点分别出现在包括315位点在内的7个位点,315位点突变占75.0%(18/24),最常见突变形式为AGC315ACC所致Ser315Thr氨基酸改变。杂交结果与测序结果符合率为93.9%(31/33),与DNA测序相比,其阳性预测值、阴性预测值、敏感度、特异度分别为90.0%、100%、100%、86.7%。结论核酸薄膜导流杂交技术能快速、简便、高效地检测临床标本中有无结核分枝杆菌,并可提示结核分枝杆菌有无异烟肼耐药性,指导临床用药。     

武汉地区耐利福平结核分枝杆菌rpoB基因突变分析

摘要：目的：探讨武汉地区结核分枝杆菌（MTB）利福平耐药株rpoB基因的突变特征。 方法：对76例MTB临床分离株包括rpoB核心区域81 bp碱基在内的428 bp碱基进行PCR测定,并进行DNA序列分析。 结果：76例临床分离MTB中利福平耐药株56例,敏感株20例。耐药株中92.9%（52/56）存在突变,共涉及10个密码子的18种突变类型。 531、526为常见突变位点,其突变率分别为57.7%(30/52)、19.2%(10/52);联合突变率为13.5%（7/52）;同时发现了509位（AGC→AGA）新的突变类型和国内少见的517位CAG缺失类型。 结论：rpoB基因突变在武汉地区利福平耐药MTB中广泛存在,并存在新的突变位点。     

RIFO杂交和限制性片段长度多态性检测耐利福平和异烟肼的结核分枝杆菌

目的评价利福平寡核苷酸探针杂交技术（RIFO杂交）和PCR-限制性片段长度多态性（PCR-RFLP）在结核分枝杆菌（MTB）耐利福平（RIF）和异烟肼（INH）快速检测中的应用价值。方法选取121株北京地区MTB菌株，分别采用RIFO杂交技术和PCR—RFLP检测RIF耐药相关基因rpoB核心区和INH耐药相关基因katG315位点突变，并对所有菌株的rpoB基因核心区进行测序验证。结果RIFO杂交检测发现，91，5％（65／71）的RIF耐药株和92．9％（52／56）的耐多药菌株（至少对RIF和INH耐药）存在rpoB基因核心区突变，而RIF敏感株中未发现突变；RIFO杂交与测序结果完全一致，测序结果中有突变的位点在RIFO杂交中均有相应的野生型杂交信号缺失；PCR-RFLP结果显示，INH耐药株中katG315突变率为60.6％（40／66）。结论rpoB基因核心区可作为RIF耐药检测的分子标志及耐多药的筛选指标；RIFO杂交技术是检测MTB耐RIF的快速、准确的实验方法，具有推广及潜在的临床应用价值；PCR—RFLP可检测出大部分INH耐药株，可作为临床INH耐药性检测的辅助手段。     

PCR-DNA测序技术检测结核分支杆菌耐多药基因突变的研究

目的应用PCR—DNA测序技术快速检测同时耐利福平和异烟肼结核分支杆菌分离株rpoB、KatG基因突变，评价其在检测结核分支杆菌耐多药性方面的价值。方法47株耐利福平和异烟肼结核分支杆菌临床分离株及30株结核分支杆菌敏感分离株用PCR—DNA测序技术分别检测rpoB、KatG基因突变。结果47株耐多药结核分支杆菌分离株中，检出43株rpoB基因突变，突变检出率为91．5％（43／47）；31株KatG基因突变，突变检出率为66．0％（31／47）；rpoB和KatG基因同时突变者31株，突变检出率为66．0％（31／47）。30株结核分支杆菌敏感株检出1株KatG基因突变。结论PCR—DNA测序技术方法敏感、准确、特异，可快速检测结核分支杆菌rpoB、KatG耐药基因突变，有利于耐多药结核分支杆菌耐药性的快速检测。     

等位基因特异性PCR法快速检测结核分枝杆菌常见耐药突变基因

摘要：目的：建立一套同步检测结核分枝杆菌(MTB)常见耐药突变基因的等位基因特异性PCR（AS-PCR）检测体系,以间接判断对利福平（RFP）与异烟肼（INH）的耐药性。 方法：用AS-PCR技术同步检测58株MTB临床分离株rpoB基因516位、526位和531位,katG基因315位及inhA基因-15位密码子突变,并与DNA测序结果进行比对。 结果：AS-PCR法对RFP耐药株检出率为95.1%（39/41）,其中rpoB基因531位、526位、516位点突变分别检出28株、10株、2株,包括531位与526位联合点突变1株;未检出突变的2株RFP耐药株经测序验证1株未发生突变、1株发生533位点突变;RFP敏感株均未检测到突变。AS-PCR法对INH耐药株检出率为86.5%（45/52）,其中katG基因315位点突变43株、inhA基因-15位点突变2株,未检出突变的7株INH耐药株经测序验证未见突变;INH敏感株均未检测到突变。 结论：等位基因特异性PCR能够快速检测MTB常见突变基因,具有较高的敏感性与特异性,快速经济、操作简便。     

PCR-SSCP快速检测结核分枝杆菌耐利福平rpoB基因的研究

目的探讨PCR—SSCP技术检测结核分枝杆菌耐利福平rpoB基因作为新的分子药敏试验方法的价值。方法应用PCR—SSCP技术检测结核分枝杆菌耐利福平rpoB基因核心区的突变，同时对片段长度为215bp的PCR扩增产物进行测序分析。受试菌为23株利福平敏感结核分枝杆菌以72．35株利福平抗性结核分枝杆菌。结果23株利福平敏感结核分枝杆菌分别用PCR—SSCP和PCR扩增片段测序均没有检测到巾0B碱基突变。而在35株利福平抗性菌株中，PCR—SSCP检测到31株与H37Rv标准株不同的带谱，PCR—SSCP检测基因突变的敏感度和特异度分别为88．6％（31／35）和100％（23／23）。对35株利福平抗性菌株PCR扩增产物进行测序分析，在其中32株中检出了基因突变。测序分析检测基因突变的敏感度和特异度分别为91．4％（32／35）和100％（23／23）。卡方检验，PCR—SSCP和PCR扩增片段测序两种方法的敏感度之间没有显著性差异（P〉0．05）。若以DNA测序为标准，则PCR—SSCP检测基因突变的准确度、敏感度和特异度分别为93．1％（54／58），96．9％（31／32）和100％（23／23）。结论PCR—SSCP检测结核分枝杆菌耐利福平rpoB基因突变可用于利福平药物敏感度的快速测定。     

PCR—SSCP快速检测结核分枝杆菌耐利福平rpoB基因的研究

目的探讨PCR—SSCP技术检测结核分枝杆菌耐利福平rpoB基因作为新的分子药敏试验方法的价值。方法应用PCR—SSCP技术检测结核分枝杆菌耐利福平rpoB基因核心区的突变，同时对片段长度为215bp的PCR扩增产物进行测序分析。受试菌为23株利福平敏感结核分枝杆菌以72．35株利福平抗性结核分枝杆菌。结果23株利福平敏感结核分枝杆菌分别用PCR—SSCP和PCR扩增片段测序均没有检测到巾0B碱基突变。而在35株利福平抗性菌株中，PCR—SSCP检测到31株与H37Rv标准株不同的带谱，PCR—SSCP检测基因突变的敏感度和特异度分别为88．6％（31／35）和100％（23／23）。对35株利福平抗性菌株PCR扩增产物进行测序分析，在其中32株中检出了基因突变。测序分析检测基因突变的敏感度和特异度分别为91．4％（32／35）和100％（23／23）。卡方检验，PCR—SSCP和PCR扩增片段测序两种方法的敏感度之间没有显著性差异（P〉0．05）。若以DNA测序为标准，则PCR—SSCP检测基因突变的准确度、敏感度和特异度分别为93．1％（54／58），96．9％（31／32）和100％（23／23）。结论PCR—SSCP检测结核分枝杆菌耐利福平rpoB基因突变可用于利福平药物敏感度的快速测定。     

首次复治肺结核病结核分枝杆菌对利福平耐药性与rpoB基因RRDR突变的研究

目的：研究分离自首次复治肺结核病患者的结核分枝杆菌（Mycobacterium tuberculosis, Mtb）临床菌株对利福平的耐药性, 及其与rpoB基因利福霉素耐药决定区（rifampicin resistance determining regions, RRDR）位点突变间的相关性。方法：收集2013年2月至2013年12月国内19家医院的首次复治肺结核患者的Mtb, 进行利福平药敏检测;使用PCR技术检测Mtb的rpoB基因RRDR位点突变。结果：来自全国12个省19家医院共141例患者的141株临床菌株获得药敏结果, 而RRDR位点测序分析显示首次复治结核患者中有53%（75/141）对利福平耐药。RRDR DNA测序共检测到72株Mtb存在rpoB耐药突变区突变, 其中71株的突变发生在RRDR, 主要突变位点分别为531位（42株）、526位（11株）和516位（10株）。有75株Mtb对利福平耐药, 其中70株（93.3%）检测到rpoB突变, 69株的突变发生在RRDR;除了2株L511P、H526N突变的Mtb对利福平敏感, 70株rpoB突变株均对利福平耐药。对于复治结核患者, 检测RRDR突变判断利福平耐药有较高的特异度。结论：首次复治肺结核病患者对利福平耐药率较高, Mtb耐利福平与rpoB基因RRDR突变密切相关, RRDR突变检测可以用于快速鉴定复治肺结核病患者的Mtb利福平耐药。     

结核分枝杆菌耐利福平基因变异株的体外最小抑菌浓度研究

目的：研究结核分枝杆菌（MTB）耐利福平临床分离株基因突变对其体外最小抑菌浓度（MIC）测定结果的影响。方法用基因芯片对临床分离的对利福平耐药的175株 MTB及对利福平敏感的48株 MTB进行耐药基因检测;并对所有标本进行利福平的 MIC测定,比较不同变异株的 MIC测定结果。结果对利福平耐药的 MTB 临床分离株基因突变以531、526、516、533位点单突变为主;531位点单突变的利福平耐药变异株 MIC 高于526位点单突变的耐药变异株,差异有统计学意义（t′＝2．2237,t′α＝2．0449,P＜0．05）;526位点单突变的利福平耐药变异株 MIC高于516位点单突变的耐药变异株,差异有统计学意义（t＝2．2056,P＝0．0329,P＜0．05）。双突变者均有较高的 MIC测定值。结论合理的基因芯片设计能检出95．0％以上的对利福平耐药 MTB变异株;对利福平耐药的 MTB变异株有不同的基因突变模式,且不同突变模式的 MTB耐药程度不同,可以为临床用药提供参考。     

结核分支杆菌临床分离株rpoB基因突变的研究

目的：了解我院结核分支杆菌耐RFP临床分离rpoB基因的突变特点。方法：利用PCR和测序试验,分析了50株结核分支杆菌临床分离株及H37Rv标准株的rpoB基因,包括核心区域的81个碱基在内的184bp的基因片段,结果：20株敏感株未发现rpoB基因的突变,30株耐药株中,28株（93.3%）出现rpoB基因的突变,突变位置集中在rpoB基因核心区域内的27个氨基酸内,531,526两个密码子的突变率为60.7%（17／28）,未发现缺失或插入突变。高浓度耐药（R250）突变位置主要在531位氨基酸,占52.4%（11／21）,低浓度耐药（R50）突变位置主要在526位氨基酸,占57.1%(4/7)。结论：rpoB基因核心序列的检测,可作为利福平耐药的指标,对临床用药有指导意义。     

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.