乙型肝炎、丁型肝炎病毒联合诊断芯片制备的初步研究

目的制备联合检测乙型肝炎病毒(HBV)、丁型肝炎病毒(HDV)基因芯片并进行杂交验证。方法利用Primer Premier 5．0分别针对HBV、HDV基因保守区域设计多对PCR引物,扩增后的产物克隆至pMD18-T载体,提取阳性克隆质粒进行测序分析鉴定。用PixSys5500芯片打印仪将PCR产物打印在氨基修饰的玻片上制备成检测芯片。样品荧光标记采用限制性显示(RD)技术,标记后进行杂交验证分析。结果序列分析表明,运用PCR技术得到的多个基因片段均属于HBV、HDV特异基因。杂交结果显示,敏感性、特异性、重复性等指标均佳。结论利用PCR扩增产物作为探针制备HBV、HDV联合诊断芯片是一种快速、简便的实用方法,有着广阔的应用前景;利用RD技术标记样品可提高多种肝炎病毒混合检测的敏感性。     

HIV检测长寡核苷酸芯片的研制和临床初步应用

目的 研制人类免疫缺陷病毒(HIV)快速筛查的寡核苷酸芯片并初步应用于临床。方法 以HIV2个基因型(包括9个亚型)的32株典型性代表株和HIV-2特有的vpx序列的保守序列为靶序列,设计长寡核苷酸探针,并制备成Oligo芯片。样品经随机特异性引物PCR标记后与芯片杂交,PCR产物同时进行测序分析。结果 1例HIV患者芯片杂交结果阳性,20名健康对照血清均为阴性。阳性标本的序列分析表明,芯片杂交结果符合测序的分型结果。结论 此芯片可初步用于检测血清HIV RNA,并对HIV基因(亚)型进行分析。     

PCR结合地高辛标记探针杂交检验乙型肝炎病毒C基因启动子

应用聚合酶链式反应结合地高辛标记探针杂交方法检测乙型肝炎病毒C基因启动(HBV,CP)。对聚合酶链反应(PCR)法扩增的HBV DNA直接测序进行DNA同源性分析。并运用探针杂交对结果进行进一步鉴定;应用PCR法扩增20份患者血清,阳性率达95％(19/20),同时运用探针杂交进行进一步检验,与PCR结果符合率达90％以上,并选择中度、重度乙肝患者血清和pGEM,7Z—HBV质粒扩增的HBV.CP各一份分别进行测序,与报告基因的同源性分别为72％、66.5％、90％。建立了特异敏感的检测乙型肝炎病毒C基因启动子(HBV.CP)的PCR方法,可用于HBV基因变异规律的研究。同时初步的研究结果表明,肝炎病人的HBV.CP区存在较多的变异,可能与病情轻重有一定的相关性。     

基因芯片技术鉴定出血性大肠杆菌O157∶H7和霍乱弧菌O139

目的:建立一种同时快速检测肠出血性大肠杆菌(EHEC)O157∶H7和霍乱弧菌O139的基因芯片,并验证该芯片的特异性和敏感性。方法:选择EHEC O157∶H7的产志贺样毒素基因stx1、stx2和β-葡糖醛酸糖苷酶基因(u idA);霍乱弧菌O139的肠毒素A亚单位(ctxA)、毒力协调菌毛A亚单位(tcpA)、糖基转移酶(glycosotransferaseLPSgt)基因序列分别设计引物和探针,反向引物用荧光素Cy3标记,探针在3′端氨基修饰。在优化的PCR和杂交反应条件下,分别进行三重PCR扩增,产物混合后与芯片进行杂交,产生特异性荧光信号,进一步筛选探针。随后将筛选出的探针制备芯片用于检测临床样本。结果:PCR产物在相应探针处均产生特异性杂交信号,临床样本检测结果表明,此芯片比常规细菌学检测方法灵敏。结论:所研制的同时定性检测EHEC O157∶H7和霍乱弧菌O139的基因芯片是特异、灵敏而且快速的,为这两种肠道致病菌感染的快速诊断提供了新的方法。     

可视化基因芯片技术检测肠道病毒方法的建立

目的建立一种能同时检测10种肠道病毒的可视化基因芯片法。方法根据公开发表的10种常见肠道病毒的序列,设计病毒引物和探针,制备肠道病毒检测基因芯片。利用多重不对称PCR法扩增样品中的病毒靶片段,标记产物与基因芯片上的探针杂交,经清洗、可视化显色后进行结果分析。在优化的RT-PCR体系、杂交反应和可视化检测条件下,评价芯片的特异性、灵敏度和重复性。结果本研究共筛选出2对通用引物、3对特异性引物和1条肠道病毒属通用探针、9条特异性检测探针。该芯片具有较好的特异性和重复性,可检测出不低于102拷贝/μl的体外转录RNA。30例临床标本的芯片检测结果与荧光PCR法一致。结论本研究所建立的方法具有高通量、高特异性、高灵敏度等特点,因此在临床上具有潜在的应用前景,可以为肠道病毒诊断提供实验室依据。     

可视化基因芯片技术检测肠道病毒方法的建立

目的建立一种能同时检测10种肠道病毒的可视化基因芯片法。方法根据公开发表的10种常见肠道病毒的序列,设计病毒引物和探针,制备肠道病毒检测基因芯片。利用多重不对称PCR法扩增样品中的病毒靶片段,标记产物与基因芯片上的探针杂交,经清洗、可视化显色后进行结果分析。在优化的RT-PCR体系、杂交反应和可视化检测条件下,评价芯片的特异性、灵敏度和重复性。结果本研究共筛选出2对通用引物、3对特异性引物和1条肠道病毒属通用探针、9条特异性检测探针。该芯片具有较好的特异性和重复性,可检测出不低于102拷贝/μl的体外转录RNA。30例临床标本的芯片检测结果与荧光PCR法一致。结论本研究所建立的方法具有高通量、高特异性、高灵敏度等特点,因此在临床上具有潜在的应用前景,可以为肠道病毒诊断提供实验室依据。     

腹泻病毒化学发光基因芯片检测方法的建立和验证

目的 建立一种化学发光基因芯片检测方法,实现7种腹泻病毒,A组轮状病毒、B组轮状病毒、Ⅰ型诺如病毒、Ⅱ型诺如病毒、札如病毒、星状病毒和肠道腺病毒的快速、准确检测.方法 选择7种病毒特异性基因的保守区,设计引物与探针,制备寡核苷酸基因芯片.将多重实时荧光PCR(RT-PCR)扩增产物与带有特异性探针的芯片杂交,经洗涤、化学发光检测后进行结果分析.在优化的RT-PCR体系、杂交条件和化学发光检测条件下,评价芯片的灵敏度、重复性和特异性.结果 研制的基因芯片具有良好的特异性和灵敏度,检测体外转录RNA参考品的最低检测限为3×103拷贝/反应,检测临床样本的灵敏度为95.2％、特异性为92.1％、符合率为95.1％.结论 建立了一种基于化学发光基因芯片的腹泻病毒检测方法,此法能快速、灵敏、特异地检测和鉴别7种腹泻病毒,具有较好的应用前景.     

多聚酶链式反应与酶联免疫吸附试验检测丙型肝炎病毒感染的比较

本文报告了用逆转录套式多聚酶链式反应(RT nested PCR)和酶联免疫吸附试验(ELISA)检测血清中HCV—RNA和抗—HCV.用军科院丙肝抗体酶免试剂盒检测了2086份血清,病区和门诊病人抗—HCV阳性率为13.4%,献血员为2.2%,一些高危人群HCV感染率比较高.用军科院和肝研所两个单位的丙肝抗体酶免疫试剂盒同时检测100份血清,抗—HCV总符合率为95.0%.用PCR法检测了34份抗—HCV阳性和20份抗—HCV阴性血清,阳性符合率为70.6%,总符合率为79.6%.PCR与ELISA法检测HCV感染各有长短,目前仍需找到一种敏感、特异、简便、快速的诊断方法,以适应临床需要.     

人类致病病毒属水平基因筛查芯片的制备及其在黄病毒属检测中的初步应用

目的：制备人类致病病毒属水平高通量筛查用基因芯片，并通过检测黄病毒属病毒初步验证其筛查效果。方法：利用Clustal W和BLAST软件设计针对人类致病病毒的属特异性寡核苷酸探针，对探针浓度、杂交温度、杂交时间、不同杂交液及杂交后芯片的洗涤方法进行优化。分别用特异或随机PCR方法从病毒感染的细胞培养上清中扩增病毒核酸，在扩增过程中掺入aa-dUTP并进一步偶联cy5或cy3荧光素进行标记，随后与基因芯片进行杂交，并通过荧光扫描仪对杂交结果进行分析，然后分析检测的特异性和敏感性验证基因芯片的筛查效果。结果：共设计了针对人类医学病毒的1090条寡核苷酸探针及其他阳性、阴性对照探针，其中黄病毒属共46条探针，Tm值为77，67～83．53℃，通过随机或特异PCR扩增8株黄病毒属病毒核酸，在42℃、过夜杂交的条件下，通过基因芯片方法均获得了黄病毒属特异性杂交信号，与其他属病毒之间没有非特异性交叉反应。结论：所制备的基因芯片可用于黄病毒属病毒的快速筛查，本研究为进一步建立大规模病毒性病原体的高通量筛查与鉴定技术平台提供了实验依据。     

基因芯片技术鉴定出血性大肠杆菌O157:H7和霍乱弧菌O139

目的：建立一种同时快速检测肠出血性大肠杆菌（EHEC）O157：H7和霍乱弧菌O139的基因芯片,并验证该芯片的特异性和敏感性.方法：选择EHEC O157：H7的产志贺样毒素基因stx1、stx2和β-葡糖醛酸糖苷酶基因（uidA）;霍乱弧菌O139的肠毒素A亚单位（ctxA）、毒力协调菌毛A亚单位（tcpA）、糖基转移酶（glycosotransferase LPSgt）基因序列分别设计引物和探针,反向引物用荧光素Cy3标记,探针在3′端氨基修饰.在优化的PCR和杂交反应条件下,分别进行三重PCR扩增,产物混合后与芯片进行杂交,产生特异性荧光信号,进一步筛选探针.随后将筛选出的探针制备芯片用于检测临床样本.结果：PCR产物在相应探针处均产生特异性杂交信号,临床样本检测结果表明,此芯片比常规细菌学检测方法灵敏.结论：所研制的同时定性检测EHEC O157：H7和霍乱弧菌O139的基因芯片是特异、灵敏而且快速的,为这两种肠道致病菌感染的快速诊断提供了新的方法.     

Stress MR imaging for evaluation of popliteal artery entrapment

The popliteal artery entrapment (PAE) syndrome has been recognized as a cause of arterial occlusion in young people. It is the result of an anomaly of the relationship between the popliteal artery and the gastrocnemius muscle. Eight young healthy volunteers (16 legs) and six patients (10 legs) with suspected PAE underwent magnetic resonance (MR) imaging. Gradient-echo images were obtained in axial planes with the leg at rest and during active plantar flexion against resistance. Imaging at rest allowed identification of PAE signs in only one leg, which had an anomalous medial course of the popliteal artery. In the other cases, only the stress technique was able to show signal loss in the popliteal artery due to muscular compression (two legs) or the presence of accessory muscle slip around the vessel (two legs), as confirmed at surgery. MR imaging is therefore a useful technique for the diagnosis of PAE because of its capability of combining information obtainable with other modalities.     

MR imaging of muscle and tender points in fibromyalgia

Fibromyalgia is a syndrome manifested by chronic, diffuse muscu-loskeletal aching and soreness, palpable muscle tender points, and other symptoms. Standardized clinical diagnostic criteria have recently been developed. Skeletal muscle has been postulated as the end organ in this disease. Biochemical, histologic, electromyographic, and conventional radiographic studies have demonstrated no definitive abnormality. This study sought to establish whether magnetic resonance (MR) imaging could demonstrate any abnormality in these patients. Eighteen patients were entered in the study, 14 of whom were able to complete their examinations. T1 -weighted, T2-weighted, gradient-echo, and STIR (short-tau inversion-recovery) sequences were performed in all patients, with selected patients examined with T1weighted, gadopentetate dimeglu-mine-enhanced sequences. The trapezius and suboccipital regions were imaged in patients who, clinically, had active fibro-myalgia. No abnormalities could be detected. The authors conclude that the conventional MR imaging used in this study was unable to depict any primary skeletal muscle abnormality in fibromyalgia.     

MR angiography of congenital heart disease: Value of segmented two-dimensional inflow technique and maximum-intensity-projection display

Magnetic resonance (MR) angiography of the cardiovascular system was evaluated in 41 patients with congenital heart disease by using a two-dimensional (2D) inflow technique based on a magnetization-prepared gradient-echo pulse sequence with segmented k-space data acquisition and electrocardiographic gating at 0.5 T. Inversion and saturation prepulses were used to suppress stationary tissue and enhance intravascular signal. Presaturation slabs were applied where certain vascular structures had to be suppressed. Sequence parameters were optimized by evaluating signal intensity and contrast characteristics for various flip angles and inversion and saturation delay times. The heart and intrathoracic vasculature were encompassed with 40–50 overlapping sections. Both 2D angiograms and maximum-intensity-projection images were evaluated. Combining data sets acquired in the sagittal and transverse orientations provided the most satisfactory information about the pulmonary arteries. The highest signal-to-noise ratios were obtained with a flip angle of 65° and short prepulse delay times. Two-dimensional MR angiography can provide useful diagnostic information but requires a thorough understanding of in-plane and hemodynamically induced signal intensity changes.     

MR imaging of disorders of the brachial plexus

The authors evaluated 64 consecutive patients with suspected brachial plexus (BP) abnormalities of diverse cause with magnetic resonance (MR) imaging, using the body coil and a standardized protocol. Of the 43 patients for whom follow-up was available, 25 were suspected of having neoplastic involvement of the BP, nine had sustained injuries, and nine presented with BP symptoms of uncertain cause. MR imaging was 63% sensitive, 100% specific, and 77% accurate in demonstrating the abnormality in this diverse patient population. When patients with neoplastic and traumatic disorders were considered separately, sensitivity increased to 81%, accuracy to 88%, and specificity remained unchanged. In the patients with a clinical diagnosis of idiopathic or viral plexitis, the MR imaging findings were normal, serving to exclude other structural abnormalities. It is concluded that MR imaging is valuable in the assessment of a wide range of BP disorders.     

MR imaging characteristics of noncancerous lesions of the prostate.

Radical prostatectomy specimens from 53 men with clinical stage A or B prostate cancer were retrospectively reviewed and compared with correlative axial T2-weighted magnetic resonance (MR) images obtained just before surgery. Non-cancerous lesions were evaluated for signal intensity and location. Focal high-signal-intensity areas (n = 72) were present in 81% of patients. The 26% of lesions seen in the central gland all correlated with cystic atrophy. Of the 53 lesions seen in the peripheral prostate, 47 (89%) were cystic atrophy without associated cancer, four (7.5%) cystic atrophy with cancer, and two (3.8%) focal inflammation. Focal low-signal-intensity areas (n = 42) were present in 60% of patients. Of the 31% of lesions in the central prostate, one-fifth correlated with benign prostatic hyperplasia (BPH) and four-fifths with fibrous tissue. Of the 69% of peripheral lesions, 83% corresponded to fibrous tissue, 10% to BPH, and 7% to normal tissue. Mixed lesions (n = 42) were present in 64% of patients; 86% of these were located centrally and 14% peripherally. All mixed central lesions were BPH; the peripheral lesions were areas of combined cystic atrophy and fibrosis. BPH of low or mixed signal intensity can extend into the peripheral prostate and mimic cancer. High-intensity cystic atrophy associated with cancer can mimic normal tissue.     

肾细胞癌的MRI研究进展

肾细胞癌是最常见的成人肾脏恶性肿瘤。近年来，多种功能MRI成像技术（如扩散加权成像、灌注加权成像等）、多参数MRI联合分析以及影像组学等新兴影像处理技术被证实在肾细胞癌的诊断中具有较大的价值。目前，研究热点多集中于良恶性肿瘤的鉴别、组织学亚型的区分、肿瘤分期、预测核分级及判断预后。就MRI新技术及图像处理技术在肾细胞癌中的研究进展予以综述。     

Dynamic contrast-enhanced MR imaging assessment of vascularized free fibular grafts

Magnetic resonance (MR) imaging may be a noninvasive method for assessing perfusion of vascularized bone grafts placed for treatment of avascular necrosis. One proximal femur of seven beagles was devascularized, with insertion of a vascularized fibular graft. MR imaging at 1 week (seven dogs) and 6 weeks (five dogs) after surgery included pre- and postcontrast spin-echo sequences, unenhanced twodimensional time-of-flight (TOF) vascular imaging, and dynamic gradient-echo imaging during infusion of gadolinium. Relative signal intensity values of selected regions obtained from the dynamic gradientecho images were plotted as percent enhancement versus time. In the operated hip, MR imaging did not show enhancement in six of seven femoral heads and greater trochanters at 1 week after surgery, with similar results after 6 weeks. MR imaging of fibular grafts 6 weeks after surgery showed an initial rapid increase in enhancement and a subsequent slower increase in five of five dogs, although no enhancement was seen in six of seven dogs at 1 week. These findings contrasted with a rapid initial increase in enhancement followed by slow decline in non-operated hips. Two-dimensional TOP imaging did not show the vascular pedicle of the graft in any dog. Findings of radionuclide bone scanning performed 1 week after surgery were consistent with devascularization of the operated femur and fibular graft. However, tetracycline distribution and histologic findings confirmed the viability of five of five grafts within the devascularized femurs 6 weeks after surgery. Thus, dynamic contrast-enhanced MR imaging at 6 weeks after surgery is valuable for assessing vascular bone graft perfusion, while similar imaging at 1 week may suggest otherwise.     

Influence of in vivo copper on MR images of the liver in rats

To investigate the effects of in vivo copper on magnetic resonance (MR) images, the authors studied Long-Evans cinnamon rats, which develop hepatitis and hepatocellular carcinoma as a result of abnormal copper metabolism. The livers of the rats were imaged before hepatitis developed; the absence of hepatic disease was confirmed histopathologically. The copper that accumulated in the liver of the rats was thought to exist in the form of divalent ions, which were suspected of reducing the T1 and T2 of neighboring protons. However, the signal intensities of the liver on T1- and T2*-weighted images did not change, suggesting that in vivo copper, even when accumulated abnormally, does not influence the signal intensity of MR images.     

Techniques for high-resolution MR imaging of atherosclerotic plaque

Atherosclerotic cardiovascular disease is the most common cause of death in the United States. Investigation of atherosclerotic plaque morphology and composition is important because the findings may be useful in predicting prognosis or response to therapy. This study presents high-resolution magnetic resonance (MR) imaging techniques developed on a 1.5-T whole-body imager with a custom-built surface coil, for characterizing the composition and morphology of plaque removed at carotid endarterectomy. The initial comparison of MR imaging and histologic results showed good correlation. In conjunction with MR angiography, these techniques could be used in in vivo imaging to define the size, location, and contents of atherosclerotic plaque at the carotid bifurcation.     

Dynamic MRI of the gallbladder lesions: Differentiation of benign from malignant

Forty-nine pathologically proven gallbladder lesions were evaluated in 45 patients using dynamic MRI with a spoiled gradient pulse sequence (SPGR), to access the ability of this technique to differentiate benign from malignant gallbladder lesions. The studies were reviewed retrospectively. Signal intensity of the lesions were measured. Twenty-one malignant and 28 benign lesions were classified into three categories: polypoid, diffuse wall thickening, and exophytic. Early and delayed enhancement patterns were evaluated. For the polypoid masses, malignant lesions (n = 9) demonstrated early and prolonged enhancements, whereas benign lesions (n = 14) had early enhancement with subsequent washout (P < .05). For diffuse gallbladder wall thickening, malignant lesions (n = 6) demonstrated early and prolonged enhancement and benign lesions (n = 14) showed relatively slow, prolonged enhancement (P < .05). The exophytic masses (n = 6) all were malignant and demonstrated early and prolonged enhancement. Dynamic MRI can help differentiate benign from malignant gallbladder lesions.