PCR操作中的几点体会

多聚链酶反应(Polymerase chain relrion PCR)是将特异的核苷酸序列在体外进行多次扩增的一种新技术,近年来已广泛用于病原微生物检测、遗传病及肿瘤的诊断等生物医学领域。但PCR在实际应用中,仍存在一些问题,需不断总结并加以改进。     

重要病原微生物研究进展及展望

目的 综述国内外重要病原微生物的研究现状和发展动态,提出未来军队病原微生物学研究的发展方向.方法 采用情报调研方法,检索和综述近5年来国内外在一些重要病毒(如肝炎病毒、人类免疫缺陷病毒、流感病毒、脑炎病毒、出血热病毒)和细菌(如结核分枝杆菌、2型猪链球菌、鼠疫耶尔森菌、炭疽杆菌和幽门螺杆菌)及其所致传染病研究领域的研究进展.结果 病原微生物引起的各种传染病是目前威胁人类健康与生命的最重要的一类疾病.特别是近30年来,一些"新现传染病"和"再现传染病"造成的严重危害受到广泛关注.随着人类对病原微生物认识的不断加深,对付病原微生物的手段也不断完善.结论 "十二五"期间,军队医学微生物学领域应努力培养和造就一支具有较强创新能力的科技人才队伍,建设和完善一批功能先进配套的科研基地和技术平台,组织申报和落实重大项目,取得一批具有原始创新和重要应用前景的科研成果,提高科技成果转化率和科技进步贡献率,力争在若干重大前沿、主流领域的研究中取得重要进展和突破.     

脂血对实时荧光定量PCR实验结果影响的研究

荧光定量PCR是聚合酶链反应（polymerase chain reaction,PCR）中的一种,因其方法学的高灵敏度、高特异性、简便、快速等特点,目前在临床上有了广泛的应用,并且在病原微生物领域中显示出了巨大的应用价值和广阔的发展前景。     

女性生殖道病原微生物感染及构成分析

目的：探讨女性生殖系统炎症及其病原微生物的感染状况及构成变化，为防治女性生殖系统炎症提供参考依据。方法：对妇科门诊初诊为生殖道炎4692例，用病原培养法、抗原或抗体免疫检测法及显微镜进行病原学检测，并对其病原微生物构成进行分析。结果：10岁以下儿童组链球菌的检出率最高(36．0％)；50岁以上组念珠菌感染率最高(26．8％)，其次是假单胞菌(10．7％)；其他病原微生物以21～30岁和31～40岁两组检出率最高。病原微生物构成：性传播疾病(STD)支原体感染率60．8％，念珠菌、金黄色葡萄球菌和表皮葡萄球菌分别为20．2％、17．4％和13．5％。合并2种病原微生物感染者1361例(36．6％)，合并3种病原微生物感染者624例(16．8％)，合并4种病原微生物感染者162例(4．4％)。结论：育龄妇女为各种病原微生物的主要被感染者，应重点检测和防治；支原体、念珠菌和葡萄球菌感染已成为女性生殖道炎的主要致病微生物。     

抗病毒天然免疫反应中RIG-Ⅰ样受体调控机制新进展

宿主免疫细胞和相关的组织细胞能够通过表达病原模式识别受体（pattern recognition receptor,PRR）即Toll样受体（Toll-like receptor,TLR）、核苷酸寡聚化域样受体（NOD-like receptor,NLR）、维A酸诱导基因Ⅰ样受体（RIG-Ⅰ-like receptor,RLR）检测病毒及其他病原微生物,并将感染信号级联放大,诱发抗病毒天然免疫反应。由于PRR成员识别病原的特异性和机制各具特色,使有关PRR的鉴定及其诱发天然免疫反应的分子机制研究更加具有挑战性,特别是细胞质病原识别受体——RLR引发的信号通路研究已经成为细胞生物学与免疫学领域的热点之一。最新研究发现,泛素化、去泛素化及干扰素刺激基因（interferon stimulating gene,ISG）化等翻译后修饰对RLR介导的抗病毒天然免疫反应具有重要调控作用,成为许多病毒逃逸机体防御系统的主要分子机制。并且最近研究发现了一个新的RLR信号通路中抗病毒免疫分子STING（也称为MITA/MPYS/ERIS）,为揭示复杂的抗病毒天然免疫反应提供了新思路。     

DNA电化学生物传感器检测病原微生物的应用研究进展

病原微生物的快速检测对于疫情防控具有重要意义。与传统检测方法相比,DNA电化学生物传感器在检测度、灵敏度、检测成本与便携性等方面有诸多优势。该文综述了DNA电化学生物传感器的工作原理及其在病原体检测中的应用,重点阐述了核酸四面体结构探针和新型纳米材料在DNA电化学生物传感器中的最新进展,以及DNA电化学生物传感器检测技术在病原体现场快速检测中面临的挑战和发展趋势。     

痕量微生物快速检测技术

痕量微生物快速检测技术结合流式细胞术和图像细胞术的功能,能够实现细胞微生物的快速检测、荧光信号量化与微生物形态的可视化,其对微生物的快速准确计数,在食品、饮用水等的质量安全检测中可以发挥重要作用,对于保证居民日常生活及身体健康具有重要实际意义。该文介绍了当前几种常用的微生物检测方法,重点阐述了痕量微生物快速检测技术的应用及优缺点,并对该技术在微生物检测方面的应用前景及发展趋势进行了展望。     

CRDS呼吸分析技术及其临床应用研究

呼吸分析(Breath Analysis)是一种新颖的非侵入性的疾病诊断和代谢监测技术,对实现重大慢性疾病的无创检测和筛查有着重要意义。但目前呼吸分析研究领域的发展主要存在两大挑战:一是对确定的呼吸生物标记物和现有的临检参数缺乏定量相关性,需深入研究以实现临床诊断;二是许多疾病的生物标记物还有待研究,需要发现用于诊断的新标记物。这都需要一种高灵敏的实时在线分析技术,在极短的时间内完成大量呼吸样品研究。相比质谱技术,光腔衰荡光谱(CRDS)呼吸分析技术具有实时、低成本、无序样本前处理及可用于床旁监测等优点,是呼吸气体分析领域的新秀。目前本课题组已成功研制出便携式CRDS呼吸丙酮分析仪,其体积小、所需气量少,响应时间为1 s,测量时间约为1 min,探测极限可达50 ppb,并已在糖尿病无创诊断和动态监测领域开展了大量地临床试验。同时该仪器为平台仪器,由于不同分子对应不同波长的光谱指纹,通过选择合适波长的激光器及对应的高反射镜,就能开展其他重大疾病生物标记物的研究,建立用于肺癌、乳腺癌及慢性肾病等重大疾病诊断呼吸标记物数据库,推动呼吸分析技术在临床医学研究领域的应用。     

一种可用于癌症检测的激光拉曼光谱仪

激光拉曼光谱技术在医学、生物化学、生物物理学 ,分子生物学领域得到广泛应用。拉曼光谱可从分子水平检测癌症患者血清中代谢产物的变异。本文介绍一种自行研制的用于癌症检测的激光拉曼光谱仪。它主要由 82 2型氩离子激光器和ＧＤＳ5 0 2光栅双单色仪组成 ,具有输出功率大 (单线输出 2Ｗ)分辨率高等特点 ,杂散光达 10 -13 。用该仪器对10 70例癌患者血清拉曼光谱检测 ,与临床对照符合率 86 % ;并行ＣＡＲＳ光谱测试 ,观察到相干反斯托克斯拉曼光谱。一种可用于癌症检测的激光拉曼光谱仪@苗春安$中国科学院长春光学精密机械与物理研究所!1…     

脑氧饱和度监测在大型手术中的临床应用进展

近红外光谱技术能对脑氧饱和度进行连续、无创伤、灵敏的监测,其操作简易,能为临床诊疗提供重要诊疗依据。近年,利用反射性近红外光谱技术检测人脑组织氧饱和度（rSO2）在心脏手术、胸科手术、神经外科手术等非心脏手术、低温、低血压和重症监护等领域得到迅速发展。我们就近红外光谱技术监测脑氧饱和度在大型手术中的临床应用进展进行综述。 1脑氧饱和度监测在心血管手术中的应用     

Improved sensitivity to overlapping multiplet signals in in vivo proton spectroscopy using a multiecho volume selective (CPRESS) experiment

A method for volume selective proton spectroscopy is presented based on a multiecho sequence with short refocusing interval tcp. It is demonstrated, that by appropriate choice of tcp on the order of 4–6 ms, signals from overlapping multiplets like the glutamine and glutamate (Glu/Gln) resonances in spectra of the human brain are considerably increased compared with a conventional PRESS volume selection scheme. Thus proton spectra from J-coupled multiplet signals can be acquired with TE on the order of 20–30 ms avoiding the baseline problems arising at shorter echo times due to broad resonances. This allows to selectively acquire spectra from substances with longer T₂ without the confounding effects from J-coupling occurring in conventional volume selection techniques.     

肝脏3.0 T质子MRS谱线后处理评估者间和评估者自身可重复性

目的探讨在体肝脏3.0T质子磁共振波谱(MRS)谱线用SAGE软件Spectrum后处理方法处理评估者间和评估者自身可重复性。资料与方法19例自愿者,使用GESigna Excite HD3.0T超导磁共振扫描仪,8通道相控阵线圈,单体素点分辨波谱分析法(PRESS)序列。采集参数为TR1500ms,TE30ms,信号采集次数(NSA)64次,感兴趣容积(VOI)2cm×2cm×2cm,共获得19条谱线。采用SAGE软件包,Spectrum后处理方法,测量者1测量2次和测量者2测量1次,经过5Hz高斯窗函数截趾(Apodization)处理,傅立叶变换,谱线自动基线校正,谱线自动相位校正,手动确定4.7ppm水和1.3ppm脂肪峰亚甲基峰位置,拟合积分共得到57组水峰和脂肪峰峰高及峰下面积。(1)计算评估者间和评估者自身等级相关系数。(2)采用多组配对样本秩和检验(FriedmanTest)比较三组的水峰和脂肪峰峰高及峰下面积差异是否存在统计学意义。结果(1)测量者1前后2次测量结果脂质峰峰高及峰下面积相关系数分别为0.981(P<0.000)、0.989(P<0.000),水峰峰高及峰下面积相关系数分别为1...     

年龄对肝脏3.0 T质子MRS抑水的影响

目的探讨年龄对在体肝脏3.0T质子磁共振波谱(MRS)预扫描抑水效果的影响。资料与方法 44名自愿者,使用GE Signa Excite HD 3.0 T超导磁共振扫描仪,8通道腹部相控阵线圈,单体素点分辨波谱(PRESS)序列采集。参数为TR1500ms,TE30ms,信号采集次数(NSA)64次,感兴趣容积(VOI)2cm×2cm×2cm。采用SAGE进行后处理分析得到代谢物峰高及峰下面积。以水峰为内参照对脂质峰高度及面积进行标准化。(1)把受检者按是否年龄<44岁分为两组,比较两组受检者一般情况包括身高、体重、体重指数(BMI);波谱预扫描匀场效果和抑水效果;脂质峰和水峰的峰高及峰下面积等计量指标是否存在统计学差异,采用两独立样本的t检验,数据为非正态分布时采用两独立样本秩和检验。(2)并计算年龄与各上述指标相关系数,数据为非正态分布时计算等级相关系数。结果 (1)年龄较轻组较年龄较长组的平均BMI较小[分别为(22.0±3.5)kg/m2,(24.7±2.7)kg/m2,t=-2.793,P=0.008],Lip2峰高[两组最小值-最大值分别为14099-634600,34480-1752600;中位...     

Dual interleaved 1H and proton-decoupled-31P in Vivo chemical shift imaging of human brain

A technique is demonstrated to obtain interleaved proton (¹H) and ¹H-decoupled phosphorus (³¹P) spectra of human brain using 2D CSI. A modified commercial full-body imager and a dual-tuned birdcage head-coil were employed. Because proton relaxation times are shorter than those of phosphorus, TR(¹H) can be chosen to be shorter than TR(³¹P), thus permitting a ¹H acquisition to be inserted in each ³¹P cycle. The scheme results in significant time savings as both CSI data sets are obtained concurrently with patient loading, coil tuningi, shimming, and imaging needed to be done only once.     

Reproducibility of high spatial resolution proton magnetic resonance spectroscopic imaging in the human brain

The application of proton (¹H) magnetic resonance spectroscopic imaging (MRSI) allows for noninvasive, localized analyses of brain biochemistry; however, minimal work has been devoted to the evaluation of ¹H MRSI reproducibility. This study examined the reproducibility of ¹H MRSI from five normal subjects on two occasions, separated by 10 days. Reproducibility of the MR signal was evaluated in the context of automated shimming, automated processing, and accurate subject repositioning. Reliability measures for physicochemical indices (choline moieties, creatine, N-acetylaspartate, and myo-inositol) were moderately concordant across repeat studies. Gain variation and repositioning results were excellent. It has been concluded that ¹H MRSI reproducibility is adequate for serial studies of brain metabolism.     

Comparison of localized proton NMR signals of skeletal muscle and fat tissue in vivo: Two lipid compartments in muscle tissue

In vivo ¹H NMR spectra of small volumes-of-interest (VOI) were localized in human soleus muscle (8 ml) and compared with volume selective spectra of subcutaneous fat tissue and femoral yellow bone marrow (2 ml). All examinations were performed by the double spin echo (PRESS) localization technique. To provide comparability, spectra of different tissues were recorded using identical sequence timing. Clearly improved resolution of the lipid signals of muscle tissue was obtained using long echo times TE > 200 ms. The spectra of muscle tissue exhibit lipid signals that stem from two compartments with a difference of their resonance frequencies of about 0.2 ppm (Larmor frequency difference 12-13 Hz at 1.5 T). The existence of two fatty acid compartments is supported by measurements of the relaxation times and line shape analysis. Both compartments contain fatty acids or triglycerides with similar composition. Probably one compartment corresponds to fat cells within muscle tissue, the other compartment with lower Larmor frequency is located within muscle cells.     

Optical spectroscopy and imaging for early lung cancer detection: a review

Over the last 15 years, optical spectroscopy and imaging has been intensively studied to improve the detection and localization of early lung cancer. Autofluorescence bronchoscopy (AFB) is the most successfully developed technique and has significantly improved the detection sensitivity of early lung cancer. In this review, the optical principles behind white-light and autofluorescence bronchoscopy, as well as the role of AFB in the diagnosis of early lung cancer and the overall management of patients with early lung cancer are discussed. Other newest development such as Raman spectroscopy and simultaneous imaging and spectroscopy measurements are also highlighted.     

健康成人脑代射物浓度以及比值的1HMRS研究

目的探讨脑内代谢物及其比值与年龄变化的关系.材料和方法正常健康志愿者51名,无神经系统疾病,分为中、青年组30名(年龄18～45岁)和老年组21名(年龄62～78岁).用GE 1.5T Signa超导磁共振扫描仪,采用定点分辨波谱序列(PRESS)对额叶、颞叶、枕叶以及海马区的NAA、tCr、Cho和MI测定并计算NAA/Cr、Cho/Cr、MI/Cr的比值.结果中青年组和老年组之间上述四个部位的脑内代谢物的浓度以及其比值无明显差异.结论脑内额叶、颞叶、枕叶和海马区的NAA、Cr、Cho和MI浓度及其比值与年龄无关.     

Anomalous Transverse Relaxation in 1H Spectroscopy in Human Brain at 4 Tesla

Longitudinal (T₁) and apparent transverse relaxation times (T₂) of choline-containing compounds (Cho), creatine/phospho-creatine (Cr/PCr), and N-acetyl aspartate (NAA) were measured in vivo in human brain at 4 Tesla. Measurements were performed using a water suppressed stimulated echo pulse sequence with complete outside volume presaturation to improve volume localization at short echo times. T₁-values of Cho (1.2 ± 0.1 s), Cr (1.6 ± 0.3 s), and NAA (1.6 ± 0.2 s) at 4 Tesla in occipital brain were only slightly larger than those reported in the literature at 1.5 Tesla. Thus, TR will not adversely affect the expected enhancement of signal-to-noise at 4 Tesla. Surprisingly, apparent T₂-values of Cho (142 ± 34 ms), Cr (140 ± 13 ms), and NAA (185 ± 24 ms) at 4 Tesla were significantly smaller than those at 1.5 Tesla and further decreased when increasing the mixing interval TM. Potential contributing factors, such as diffusion in local susceptibility related gradients, dipolar relaxation due to intracellular paramagnetic substances and motion effects are discussed. The results suggest that short echo time spectroscopy is advantageous to maintain signal to noise at 4 Tesla.