聚合酶链法检测莱姆病患者尿液中伯氏疏螺旋体DNA(附17例报道)

目的:评估聚合酶链反应(PCR)检测莱姆病患者尿液中伯氏疏螺旋体DNA的诊断价值。方法:2004至2006年来自新疆莱姆病自然疫源地临床疑似莱姆病患者17例(均有蜱暴露史和莱姆病临床证据者)和6例以前确诊并治疗后完全恢复的莱姆病患者,共23例为病例组;另选择25例非疑似莱姆病患者为对照组。应用PCR方法检测尿液中伯氏疏螺旋体DNA,采用间接免疫荧光(IFA)检测血清伯氏疏螺旋体抗体。结果:①17例莱姆病患者临床表现呈多系统损害。12/17例(70.59%)为神经型(周围神经病3例,其中1例合并下肢瘀积性皮炎;游走性红斑并发莱姆脑病2例;脑膜脑炎2例;脑炎2例;脑膜炎、脑干炎和脊髓炎各1例);流感样症状2例,其中伴皮疹1例;余心脏型、精神障碍和肝病型各1例。②PCR伯氏疏螺旋体DNA检测阳性17/17例(总阳性率100%)。4例血清IFA试验伯氏疏螺旋体抗体阳性。以前确诊并治疗者6例及对照组25例尿PCR伯氏疏螺旋体DNA和血清IFA试验伯氏疏螺旋体抗体均阴性。③10例完成抗生素治疗后3个月复查,9例PCR伯氏疏螺旋体DNA检测阴性(包括3例晚期莱姆病患者),1例2年复查时仍阳性;4例血清伯氏疏螺旋体抗体阳性者复查均阴性。④早期莱姆病多采用几周的抗生素治疗方案。结论:PCR检测尿液中伯氏疏螺旋体DNA是莱姆病一个有价值的诊断工具。     

巢式聚合酶链反应检测脑脊液中单纯疱疹病毒DNA

应用巢式聚合酶链反应(PCR)检测患者脑脊液(CSF)中单纯疱疹病毒1型(HSV-1)DNA。23例经鞘内HSV-1特异性IgG抗体检测阳性的“HSV-1型性脑炎(HSE)”中19例PCR阳性,4例阴性患者病程均超过1个月。22例IgG阴性的“散发性脑炎”中7例PCR阳性,此7例皆于发病1周内检查CSF,其中2例取材于发病当日。1周后复查7例患者,CSF PCR仍为阳性,IgG皆阴性,提示PCR适用于HSE的早期诊断。     

应用差别显示聚合酶链反应筛选及鉴定重症肌无力相关基因

目的筛选及鉴定重症肌无力（ＭＧ）相关基因。方法应用差别显示ＰＣＲ（ＤＤＰＣＲ）技术比较１例ＭＧ患者受累骨骼肌和１例意外死亡者健康骨骼肌的基因表达，并用Ｎｏｒｔｈｅｒｎｂｌｏｔ研究此基因在６例ＭＧ患者和５名健康人骨骼肌和胸腺组织中的表达情况。结果经ＤＤＰＣＲ筛选到１个与ＭＧ相关的、全长约６ｋｂ的新基因，暂命名为ＬＰＷ１；Ｎｏｒｔｈｅｒｎｂｌｏｔ显示，在所有ＭＧ患者的骨骼肌组织和增生胸腺中，ＬＰＷ１ｍＲＮＡ表达水平均低于健康人组，而在胸腺瘤组织中的表达则高于健康人组；ＬＰＷ１不是骨骼肌特异的基因，在健康人肝、脑、胃、骨骼肌、胸腺和胎肾等组织中均有表达。结论ＬＰＷ１为ＭＧ相关基因，在ＭＧ的发病机制中可能起一定作用；伴发胸腺增生的ＭＧ和伴发胸腺瘤的ＭＧ的发生机制可能有所不同。     

基因学快速检测脑脊液常见细菌感染的方法

目的 探讨基因芯片技术对于颅内感染致病菌快速检测的应用价值.方法 选择4种常见致病菌以及6种常见耐药基因的特异DNA序列设计相应的引物与探针,对30例颅内感染患者的脑脊液标本(脑脊液培养阳性12例,阴性18例)进行多重PCR扩增、基因芯片检测,进行菌种鉴定和耐药基因检测,与脑脊液细菌培养及药敏检测结果进行比较.结果 15例标本鉴定出菌种并检出耐药基因;8例未鉴定出菌种但检出细菌及耐药基因;7例未检出细菌及耐药基因.结论 基因芯片可以相对灵敏、快速地用于颅内感染患者脑脊液中致病菌检测.     

聚合酶链反应及抗体检测对单纯疱疹病毒性脑炎早期诊断的意义

通过特异性抗体（ＥＬＩＳＡ）及聚合酶链反应（ＰＣＲ）检测，３８例散发性脑炎中１８例诊断为单纯疱疹病毒性脑炎（ＨＳＥ），２０例为非ＨＳＥ的散发性脑炎（ＮＨＳＳＥ）。并与３０例其它神经系统疾病患者作三组对比性临床及实验室研究，发现ＨＳＥ在临床表现、脑脊液、头颅ＣＴ等方面有其特征性。１８例ＨＳＥ的２４份脑脊液（ＣＳＦ）标本中，ＰＣＲ阳性１８份，ＩｇＧ阳性１９份，其中１３份ＰＣＲ和ＩｇＧ皆阳性；其余两组ＰＣＲ和ＩｇＧ均阴性。ＰＣＲ和ＩｇＧ检测有可能成为ＨＳＥ常规诊断方法，ＰＣＲ于发病当天即可获得诊断。     

应用实时荧光定量聚合酶链反应结合突变阻滞形成系统进行线粒体脱氧核糖核酸A3243G突变负荷检测

目的 应用实时荧光定量PCR结合突变阻滞形成系统(RT ARMS-qPCR)技术检测线粒体DNA A3243G突变负荷,并探讨线粒体脑肌病伴高乳酸血症和脑卒中样发作(MELAS)综合征患者不同组织中A3243G突变负荷的分布情况.方法 分别构建含线粒体DNA3243位点的野生型(A)和突变型(G)序列的质粒,将这2种质粒按一定比例混合构建成13个含有不同A3243G突变负荷的标准对照,应用PCR-限制性片段多态性技术(RFLP)和RT ARMS-qPCR技术检测13个标准对照及存在A3243G突变的7例MELAS患者、1名携带者以及53名健康对照的外周血、肌肉、毛发及尿沉渣等组织的A3243G突变负荷.结果 标准对照的突变负荷经PCR-RFLP和RT ARMS-qPCR检测的结果均与预期值呈直线相关,决定系数分别为R21=0.885、R22=0.991,RT ARMS-qPCR检测的结果更接近预期值;7例MELAS患者及1名携带者不同组织的突变负荷经RT ARMS-qPCR检测的结果比经PCR-RFLP检测出的高,且RT ARMS-qPCR比PCR-RFLP更容易检测出低负荷的A3243G突变,并发现MELAS患者的肌肉、尿沉渣、头发的A3243G突变负荷均高于外周血.结论 应用RT ARMS-qPCR可以快速、灵敏、准确地检测不同组织的A3243G突变及其突变负荷.     

假肥大型肌营养不良基因突变检测方法的应用比较

目的比较假肥大型肌营养不良基因突变各种检测方法的优缺点,为不同条件下选择最佳的检测方案提供借鉴。方法分别应用18对引物多重PCR和5×4DNA微阵列对30例假肥大型肌营养不良患者进行dystrophin基因(缺失)检测分析,并结合文献中的方法进行应用比较。结果 30例假肥大型肌营养不良患者中有21例至少存在一个外显子片段缺失,占总例数的70%;9例未被检测到缺失,点30%。末检测到全部缺失的患者。PCR、DNA微阵列检测结果一致。结论对于假肥大型肌营养不良患者及携带者可选择MLPA检测缺失和重复突变,对于阴性者再利用PCR联合DHPLC结合测序检测点突变。经济方案是先选择18对引物定量PCR或DNA微阵列检测常见外显子缺失和重复突变,对于阴性者再用MLPA检测其它外显子缺失和重复突变。对于可疑胎儿产前诊断行MLPA或PCR-STR连锁分析。     

人β-神经生长因子前体基因的克隆及序列分析

目的 对人β-NGF前体基因的克隆及序列分析,为其在真核细胞中表达并获得具有神经生长因子活性的蛋白及临床应用打下基础。方法 从人血白细胞中提取基因组DNA,采用PCR技术,扩增出含前导肽及信号肽的β-NGF前体基因并与PUC18载体连接,经筛选得到重组质粒PUC18-β-NGF,经酶切及序列分析进行鉴定。结果 经序列测定与Genebank中已知序列(V01511)相比较,完全一致。结论 从人白细胞DNA中获得了人β-NGF前体基因,为神经生长因子的基因治疗提供了条件。     

纳米羟基磷灰石颗粒对巨噬细胞DNA的损伤

目的：纳米颗粒由于其特殊的尺寸效应，会对细胞内处于纳米尺寸级的RNA、DNA和蛋白质等生物大分子直接产生不良影响，使生物体受到损害。因此，从分子水平上探讨纳米羟基磷灰石颗粒对细胞DNA损伤的相关基因或蛋白的影响具有重要的科学意义。 方法：实验于2005-12/2006-12在上海生物材料研究测试中心完成。①实验材料：纳米羟基磷灰石颗粒由中科院上海硅酸盐所提供，粒径大小为30～80 nm。300 W/40 kHz的超声条件下，用含体积分数为0.1小牛血清的RPMI-1640振荡纳米颗粒，制备成终浓度为1 g/L的悬浮液，4 ℃保存1周。②实验方法：选用RT-PCR的技术，通过原代培养大鼠腹腔巨噬细胞，在分子水平上检测纳米羟基磷灰石颗粒对DNA损伤通路中P53、P21、gadd45以及HSP70这些关键蛋白转录水平的影响。 结果：通过mRNA水平的检测，发现P53、P21及HSP70的表达均随着纳米羟基磷灰石颗粒浓度的增加而升高，并在100 mg/L条件下P53、P21及HSP70有显著性表达（P < 0.05），而当纳米羟基磷灰石颗粒达到200 mg/L，只有P21的表达反而有所降低，同时各浓度的纳米羟基磷灰石颗粒对gadd45表达无明显影响（P > 0.05）。 结论：实验证实了100 mg/L 纳米羟基磷灰石颗粒可以引起DNA的明显损伤。并且当纳米羟基磷灰石颗粒浓度达到200 mg/L时，受损的DNA已不易被修复。同时，P53和HSP70可成为评价纳米羟基磷灰石颗粒安全性较为灵敏的指标。     

人β-神经生长因子前体基因的克隆及序列分析

目的 对人β-NGF前体基因的克隆及序列分析，为其在真核细胞中表达并获得具有神经生长因子活性的蛋白及临床应用打下基础。方法 从人血白细胞中提取基因组DNA，采用PCR技术，扩增出含前导肽及信号肽的β-NGF前体基因并与PUC18载体连接，经筛选得到重组质粒PUC18-β-NGF，经酶切及序列分析进行鉴定。结果 经序列测定与Genebank中已知序列(V01511)相比较，完全一致。结论 从人白细胞DNA中获得了人β-NGF前体基因，为神经生长因子的基因治疗提供了条件。     

Detection of varicella-zoster virus DNA by polymerase chain reaction in cerebrospinal fluid of patients with herpes zoster meningitis

Summary In three of five patients with herpes zoster meningitis, varicella-zoster virus (VZV) DNA was detected by the polymerase chain reaction (PCR) in the initial samples of cerebrospinal fluid. DNA fragments of group A or B, following classification of VZV strains by the size of the variable region IV of VZV genome, were found at the 7th, 10th and 24th illness day in the three positive cases: one of these cases did not have skin lesions. These results suggest that the detection of VZV DNA by PCR is useful for the diagnosis of herpes zoster meningitis, as well as for its molecular epidemiology.     

聚合酶链反应诊断单纯疱疹病毒性脑炎

用聚合酶链反应（ＰＣＲ）扩增患者脑脊液（ＣｈＦ）中病毒特异性ＤＮＡ可早期快速诊断单纯疱疹病毒性脑炎（ＨＳＥ）。１９例临床确诊的病毒性脑炎患者，经ＰＣＲ在ＣＳＦ中检出单纯疱疹病毒（ＨＳＶ）１３例，全部阳性标本均经分子杂交证实为ＨＳＶ－ＤＮＡ，而１４例其他神经疾病（ＯＮＤ）对照组均为阴性，显示了这一方法的特异性。其中７例病毒性脑炎ＣＳＦ标本分别用ＰＣＲ分子杂交和病毒分离等三种方法检测ＨＳＶ；显示ＰＣＲ最为敏感。表明ＰＣＲ技术的广泛应用将提高ＨＳＥ的早期诊断水平，指导临床正确治疗。     

Hepatitis C virus infection and myositis: a polymerase chain reaction study

Muscle biopsy tissue from a patient with chronic hepatitis, who was hepatitis C virus (HCV) positive and showed slight weakness of the right arm and leg associated with increased serum creatine kinase levels, was studied using immunocytochemical and polymerase chain reaction (PCR) techniques. Muscle biopsy showed changes compatible with an inflammatory myopathy. Immunohistochemical studies included the use of monoclonal antibodies against human T lymphocytes, macrophages, immunoglobulins, major histocompatibility complex class I molecules (MHC-I), and the neoantigens of the terminal C5b-9 complement membrane attack complex (MAC). In addition to confirming the potential importance of cytotoxic T cells and MHC-I antigen expression in inducing muscle pathology, we demonstrated MAC deposition and the presence of HCV-RNA in the muscle of our patient, suggesting that direct involvement of the virus leading to complement activation might be important in inducing muscle damage. Received: 30 November 1998 / Revised: 17 March, 29 June 1999 / Accepted: 30 June 1999     

实时荧光定量聚合酶链反应诊断面肩肱型肌营养不良

目的 建立面肩肱型肌营养不良(FSHD)的实时荧光定量PCR(FQ-PCR)检测方法.方法 常规酚-氯仿法抽提基囡组DNA,通过EcoR Ⅰ酶切及琼脂糖凝胶电泳,回收38 kb以下DNA作为模板,根据4号染色体上D424序列设计特异的引物和探针,对115例研究对象进行FQ-PCR检测,根据荧光曲线与阳性对照的比较判断结果.结果 16例已知EcoR Ⅰ片段大小的FSHD患者FQ-PCR检测结果为13例阳性,78名健康人检测结果除3例阳性外其余均为阴性,16例经临床诊断的新FSHD患者和5名高危者中分别有15例和3例检测结果为阳性.统计学分析FQ-PCR方法与传统印迹杂交方法(κ=0.765,P=0.002)及临床诊断(κ=0.844,P=0.000)之间的一致性,结果有统计学意义.结论 我们创建了以FQ-PCR技术对FSHD进行基因诊断的新方法.该方法能克服印迹杂交方法费时费力、有放射性污染的缺点,且能较好解决由于4q-10q易位及p13E-11探针结合部位缺失造成传统杂交基因诊断方法欠准确的问题,具有较好的临床应用价值.     

Study of mitochondrial DNA depletion in muscle by single-fiber polymerase chain reaction

We studied muscle biopsies from 3 children with a mitochondrial myopathy characterized histochemically by the presence of ragged-red fibers (RRF) and various numbers of cytochrome c oxidase (COX)-negative fibers. We quantitated the absolute amounts of total mitochondrial DNA (mtDNA) in isolated normal COX-positive muscle fibers and in COX-negative RRF. There was severe mtDNA depletion in all fibers from the two most severe cases. In the third case mtDNA depletion could not be established with conventional diagnostic tools, but it was documented in single COX-negative fibers; COX-positive fibers showed the same amounts of mtDNA as fibers from aged-matched controls. Our observations indicate that mtDNA single-fiber PCR quantitation is a highly sensitive and specific method for diagnosing cases with focal mtDNA depletion. This method also allows one to correlate amounts of mtDNA with histochemical phenotypes in individual fibers from patients and age-matched controls, thereby providing important information about the functional role of residual mtDNA. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1374–1381, 1998     

Toxoplasmic encephalitis of basal ganglia with tumor‐like features proven by pathogen‐specific polymerase chain reaction and direct DNA sequencing

We report a case of a young female patient who developed progressive neurological dysfunction with a ring‐enhancing tumor‐like nodule on brain magnetic resonance imaging. Urgent surgery was performed to remove the mass in the left basal ganglia. Pathological findings showed that the necrotic brain areas were accompanied by congestion, edema, discrete hemorrhage, and intestinal and perivascular lymphohistiocytic infiltration. Immunohistochemical staining results showed that Toxoplasma gondii (T. gondii) immunoreactivity was detected in both cysts and tachyzoites in these areas. The glycerol‐3‐phosphate dehydrogenase gene (B1) of T. gondii was amplified by sequence‐specific polymerase chain reaction (PCR) and the PCR products were bi‐directional Sanger sequenced. A 195 bp consensus sequence of the gene B1 was found to be 98% identical to a reference T. gondii sequence (GenBank accession No. kx270373). The final diagnosis was toxoplasmic encephalitis in the left basal ganglia. This report suggests that PCR and bi‐directional DNA sequencing of T. gondii gene might be the most convenient and rapid tools for accurate diagnosis of toxoplasmic encephalitis .     

酶联免疫斑点法和IS6110聚合酶链反应在早期诊断结核性脑膜炎中的价值

目的 探索一种敏感而特异的早期诊断结核性脑膜炎(TBM)的方法.方法 对25例TBM患者及作为对照的27例其他中枢神经系统感染性疾病患者、22例非感染性其他神经系统疾病患者同时应用酶联免疫斑点法(ELISPOT)检测脑脊液和外周血中的抗卡介苗(BCG)抗体分泌细胞数,应用PCR检测脑脊液中结核杆菌DNA的特异性重复插入片段IS6110,用酶联免疫吸附法(EUSA)检测脑脊液和外周血抗BCG抗体.结果 ELISPOT的敏感性(84.0%)明显高于IS6110PCR(75.0%)及ELISA(52.3%),三者的特异性分别为91.8%、93.7%和91.7%.TBM患者脑脊液抗BCG-IgG分泌细胞的个数在疾病早期最高,随病程的延长逐渐下降(t=-3.183,P=0.008).结论 ELISPOT对早期诊断TBM有一定的价值.     

Intractable seizure disorder associated with chronic herpes infection HSV1 detection in tissue by the polymerase chain reaction

We describe the pathological findings and report the detection of herpes simplex virus 1 (HSV1) in the brain in three patients who presented with intractable seizures. All three patients had a previous history of HSV1 encephalitis and went on to develop a medically refractory seizure disorder necessitating surgical intervention. HSV1 encephalitis was clinically diagnosed and treated at 6 months, 3 years, and 7 months and surgical resection was done at 8.5 years, 6 years, and 3 years, in cases 1, 2 and 3, respectively. Pathological examination revealed chronic encephalitis in all three cases, with microglial nodules, intraparenchymal, perivascular and meningeal lymphocytic infiltrates, and gliosis. While immunohistochemical and ultrastructural studies were negative for viral pathogens, polymerase chain reaction (PCR) analysis revealed HSV1 genome. These cases represent examples of chronic herpes encephalitis and seizure disorder with presence of viral genome in the brain long after the initial episode of treated herpes encephalitis. Received: 15 September 1997     

Screening of gene deletions by polymerase chain reaction in Japanese patients with Duchenne muscular dystrophy

Summary Gene deletions were screened in 49 Japanese Duchenne muscular dystrophy patients from 43 families, using the polymerase chain reaction. Enzymatic amplification was carried out on six regions prone to deletion. Fifteen of 43 families (33%) had gene deletions in at least one of the six regions. This frequency was almost the same as that previously reported in Caucasians. The mid-part of the dystrophin gene was the location most frequently deleted. The frequency of deletion of the region encompassing exon 45 was higher in Japanese families (18.4%) than in Caucasians.