多种一步法快速提取DNA对肺炎支原体PCR检测的影响

目的 对比不同一步法提取DNA在肺炎支原体PCR检测中的效果及应用范围,为临床标本中肺炎支原体的检测提供依据。方法 以两种经典的DNA提取一步法（ROSE和Chelex-100法）及其优化方案和商品化DNA提取试剂盒,提取肺炎支原体纯培养菌株及30份肺炎支原体阳性临床咽拭子标本DNA,分别进行普通PCR和荧光PCR检测,对比不同方法提取的DNA对PCR检测结果的影响。结果 ROSE法和Chelex-100一步法中的SDS严重影响Taq酶活性,提取物稀释100倍方可进行荧光PCR扩增,稀释10倍可用于普通PCR扩增。改良的Chelex-100一步法提取肺炎支原体纯菌DNA产量最高,且PCR扩增效果最好。对于30份临床标本来说,试剂盒法、改良Chelex-100法和水煮法的阳性率分别为100.00%（30/30）、80.00%（24/30）和43.33%（13/30）,且对相同肺炎支原体阳性标本提取的DNA,试剂盒法检测的循环阈值（Ct值）比上述两种方法分别小1.95和2.38。结论 经典一步法提取的DNA必须经稀释后方可作为扩增模板。改良的Chelex-100法操作简便,可用于纯菌培养的DNA提取。临床标本中肺炎支原体DNA提取以试剂盒法最优,改良的Chelex-100法提取的DNA也可用于临床标本中肺炎支原体的检测,但不可用于定量分析。     

从石蜡包埋的淋巴瘤组织提取DNA并定量检测EB病毒

目的 定量检测石蜡包埋的淋巴瘤组织中EB病毒（EBV）的表达,方法 对TES水浴DNA提取法进行了改良,从石蜡包埋的淋巴瘤组织中提取高质量的基因组DNA,以实时荧光定量PCR方法检测淋巴瘤组织EBV。结果在60例标本中平均提取DNA量为3．97ng（10／μm厚切片2片）,86．7％（52／60）标本DNA的纯度（260nm／280nm）≥1．6,其中50％（30／60）淋巴瘤组织EBV呈阳性表达（10^0～10^6拷贝／ngDNA）。结论 采用改良TES水浴法能获取石蜡包埋组织中高质量基因组DNA,使用该新的实验技术可有效地利用现有的标本资源进行与疾病相关科学的研究。     

一种改良的人DNA微量快速检验技术

目的 探索一种以口腔粘膜脱落细胞为材料的安全、高效、低成本、敏感性高的人DNA微量快速检测方法。方法 采用从漱口液或棉签擦拭口腔粘膜获取脱落细胞,应用混合树脂（Chelex100）煮沸沉淀法提取DNA;用PCR分别对线粒体特异性DNA片断和基因组中的特定基因进行扩增检测。结果 通过对线粒体DNA中440 bp的非编码片段和染色体基因组中220 bp的乙醛脱氢酶DNA片段进行扩增,结果显示,从漱口液脱落细胞提取的DNA中可以稳定地扩增出上述两种DNA片断。结论 建立了一种改良的人口腔粘膜脱落细胞DNA微量快速检验技术。该法取样方便,DNA样品获取量较大,一次取样可同时进行多项线粒体和基因组标志DNA片段的快速PCR检测。     

四种石蜡包埋组织DNA提取方法的比较

背景:由于在甲醛固定石蜡包埋组织的制作及保存过程中对DNA造成的损害,影响了后续的聚合酶链反应等研究.选择一种简便有效且经济实用的石蜡包埋组织DNA提取方法成为研究者们关注和亟待解决的问题.目的:比较甲醛固定石蜡包埋组织中4种提取DNA的方法对DNA质量的影响,探讨一种操作简便、污染少、经济实用的石蜡包埋组织中提取DNA的方法.方法:取手术切除的普通甲醛固定石蜡包埋的非小细胞肺癌组织标本20例,分别以二甲苯脱蜡-酚氯仿法、改良TES水浴脱蜡-酚氯仿法、试剂盒法和改良TES水浴脱蜡-试剂盒抽提DNA法提取其DNA,然后进行电泳分析、紫外分光光度计测定A260/A280比值及PCR扩增.结果与结论:改良TES水浴脱蜡-酚氯仿法和改良TES水浴脱蜡-试剂盒抽提DNA法可获得较大的DNA片段,且两种方法与试剂盒法所提取DNA的A260/A280值相比较,均有显著性意义(P<0.05),4种方法的提取效率差异无显著性意义(P>0.1),以改良TES水浴脱蜡-酚氯仿法所得DNA为模板,扩增的目的条带亮度与阳性对照相当.结果证实改良TES水浴脱蜡-酚氯仿法简便有效,所用试剂价格低廉,是一种经济实用的石蜡包埋组织DNA提取方法.     

免疫组化与原位杂交双标记技术检测EB病毒用于诊断鼻咽癌

目的探析鼻咽癌诊断中检测EB病毒更为快速及更灵敏的方法。方法选择2014年4月至2016年8月广东省四会市人民医院收治的36例鼻咽癌患者,分别采用EB病毒一抗、地高辛标记的寡核苷酸EBER探针进行免疫组化和原位杂交双重标记染色,检测鼻咽癌细胞中EB病毒的表达情况。结果免疫组化后的阳性细胞的胞膜表现为红色,原位杂交后的阳性细胞胞核表现为蓝黑色,而双阳性细胞的核与膜呈蓝红色,不仅对比鲜明而且背景非常清晰,双染成功后细胞及组织结构表现完整。结论 EBER原位杂交双标记技术检测EB病毒主要是检测病毒基因,具有极高的特异性和灵敏度,比免疫组化方法更灵敏、快速,可以帮助临床进行鼻咽癌的分期诊断及判断治疗效果。     

EB病毒BMRF1基因原核表达载体的构建

目的克隆EB病毒早期蛋白P54的编码基因BMRF1，构建原核表达载体，为相应蛋白的表达和应用奠定基础。方法以含EB病毒的B95-8细胞培养上清为模板，PER扩增出BMRF1基因。PER产物经BamH I和Xho I双酶切后克隆至原核表达载体pGEX-5T，用双酶切和DNA测序鉴定重组质粒。结果PER扩增的特异性片段长度为1233bp，以此构建的重组质粒pGEX-5T-BMRF1双酶切的片段大小与预期符合，重组克隆外源基因的测序结果与GenBank中的EB病毒BMRF1基因eDNA序列一致。结论成功构建了pGEX-5T-BMRF1原核表达载体。     

石蜡包埋淋巴组织三种微量DNA提取方法的比较

目的为寻找一种高效、简便、实用的石蜡组织中DNA提取方法，本研究比较了三种不同的DNA提取方法对DNA质量的影响。方法选取淋巴相关组织的蜡块21例。三种DNA提取方法分别是简单消化煮沸法，酚／氯仿提纯法和试剂盒法。通过提取DNA的浓度和纯度、PCR扩增保守序列pglobin等方法，比较三种DNA提取方法的优劣。结果试剂盒法提取样品OD260／OD280平均比值位于1．6～1．8之间；提纯法位于1．3—1．5之间；简单消化煮沸法位于1，2—1．3之间。三种方法中，试剂盒法提取的DNA最为稳定。以3种DNA提取方法所获取DNA为模板，PCR扩增阳性率无统计学差异。结论从实用、快速、经济的角度综合分析，简单消化煮沸法简单快速，需要的组织样品少，是一个值得推荐的石蜡组织DNA提取方法。     

石蜡包埋组织提取DNA不同条件下的PCR扩增

背景:在甲醛固定石蜡包埋组织的制作及保存过程中会对DNA造成损害,提取的DNA在用于PCR扩增时部分结果并不理想。目的:比较分析石蜡包埋组织中提取DNA应用于PCR反应的影响因素。方法:从10.0μm×2、10.0μm×4和10.0μm×5石蜡包埋食管癌组织切片中提取DNA,以0.05,0.1,0.2μg模板DNA量扩增内参基因β-actin,PCR反应循环次数分别为35,40,45次,分析影响PCR反应的因素。结果与结论:琼脂糖凝胶电泳结果显示以10.0μm×2组织切片量,PCR反应循环次数40次,PCR反应模板DNA量0.05μg扩增取得的目的基因DNA的质量最高。说明减少石蜡包埋组织用量和减少模板DNA量有助于获得高质量的PCR反应条带,且PCR反应循环次数应大于40次,小于45次,再增加循环次数,则意义不大。     

基因组DNA快速提取方法的探讨与应用

目的 探讨以硫氰酸胍作为组织细胞裂解液的可行性,并建立基因组DNA快速提取方法.方法 利用硫氰酸胍等作为裂解消化液,分别从组织、细胞和全血中提取基因组DNA.采用紫外分光光度仪检测基因组DNA含量及纯度,琼脂糖凝胶电泳检测基因组DNA的完整性,以提取的基因组DNA作为模板进行PCR扩增GAPDH基因,并用该方法检测凋亡细胞DNA ladder.结果 基因组DNA提取时间短(耗时30～50 min),基因组DNA含量85 μg/mg(组织),52 μg/1×107细胞,27 μg/ml(血液).A260 nm/A280 nm比值在1.8～1.9之间,电泳显示基因组DNA完整性好,无蛋白质和RNA污染,基因组DNA长度大于48 kb.PCR扩增出306 bp GAPDH目的 基因,肿瘤细胞经抗癌基因诱导凋亡后琼脂糖凝胶电泳可见180～200 bp典型的凋亡带.结论 硫氰酸胍可作为基因组DNA提取裂解消化液,该方法具有简便、快捷、稳定性好等特点.     

改良的石蜡包埋组织中提取高质量基因组DNA

从石蜡包埋组织中提取基因组DNA，采用聚合酶链反应（PCR）进行基因检测，在临床常用于疾病的回顾性调查和研究。目前国内外从石蜡包埋组织中提取基因组DNA，多采用经二甲苯脱蜡，该方法可以获得所需要的DNA，但整个抽提过程过长、所得的DNA量很较少，并且易出现DNA链的降解（小片段的DNA），同时在提取过程中所使用的有机溶剂二甲苯试剂易造成环境的污染和对操作者身体的伤害。为了改进从石蜡包埋组织中提取基因组DNA的方法。     

鼻咽癌组织中EBER—1表达及其临床病理意义

目的探讨EBV与鼻咽癌发生发展关系及其临床病理意义、方法应用原位杂交技术检测76例鼻咽癌石蜡切片组织中EBER-1表达及分布状况。结果正常鼻咽粘膜上皮及其单纯性增生、鳞状化生上皮和头颈部其它肿瘤EBER＿1均阴性，鼻咽癌组织中EBER-1表达率为97．4%（74／76），且在转移灶中不丢失。鼻咽异型增生上皮亦可表达EBER＿1阳性信号（15／18）。结论EBV与鼻咽癌关系十分密切，EBV可能在鼻咽上皮向恶性转化过程中起重要作用。检测EBER-1对鼻咽癌诊断与鉴别诊断有重要价值。鼻咽上皮异型增生尤其是EBER-1间性者应认为是高危癌前病变，应密切随访监测可能有利于早期发现鼻咽癌。     

Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens

We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.     

鼻咽癌病人放疗前后VCA—IgA抗体及EB病毒DNA水平变化

目的检测鼻咽癌病人放疗前、放疗1个疗程及放疗结束后血清EB病毒衣壳抗原IgA（VCA—IgA）抗体水平及EB病毒DNA（EBVDNA）载量的变化,探讨其在鼻咽癌疗效监测及预后评估中的价值。方法收集EBV阳性鼻咽癌病人放疗前、放疗1个疗程及放疗结束后的血清标本,提取外周血细胞DNA。应用ELISA试剂盒检测血清VCA—IgA抗体水平的变化,实时荧光定量PCR方法检测EBVDNA载量的变化。结果22例鼻咽癌病人3个不同放疗阶段血清VCA—IgA抗体和EBVDNA水平比较差异均有显著性（F=3．305、9．461,P〈O．05）。放疗结束后VCA-IgA抗体水平显著低于放疗前水平（t=2．081,P〈O．05）,而放疗1个疗程后与放疗前及放疗结束后相比差异均无显著性（P〉0．05）。7例病人从治疗开始至治疗结束均未检测到EBVDNA,其余15例病人EBVDNA载量放疗前与放疗1个疗程及放疗结束后相比差异均有显著性（t=2．47、4．34,P〈0．05）,而放疗1个疗程和放疗结束后相比差异无显著性（P〉0．05）。结论同时检测血清中EBV抗体及EBVDNA水平有助于鼻咽癌疗效监测及预后评估。     

Improved accuracy of detection of nasopharyngeal carcinoma by combined application of circulating Epstein-Barr virus DNA and anti-Epstein-Barr viral capsid antigen IgA antibody

BACKGROUND: Circulating Epstein-Barr viral (EBV) DNA and anti-EBV capsid antigen IgA (IgA VCA) represent two of the most sensitive peripheral blood markers of nasopharyngeal carcinoma (NPC), but direct comparative studies of these two markers are lacking. METHODS: The sensitivities and specificities of IgA-VCA and EBV DNA for diagnosis of NPC were determined in 139 new cases of NPC and 178 healthy individuals, respectively. EBV DNA was also assessed in 36 healthy family members identified as having false-positive IgA-VCA results at a screening clinic. EBV DNA was measured by a real-time quantitative PCR assay with a detection limit of 60 copies/mL. IgA-VCA was measured by semiquantitative indirect immunofluorescent method; a titer > or =1/10 was taken as positive. RESULTS: The sensitivities of EBV DNA and IgA-VCA for diagnosis of NPC were 95% (95% confidence interval, 91-98%) and 81% (73-87%), respectively. The combined marker panel had an overall sensitivity (positive result by either marker) of 99%. The concentrations of both markers showed dependence on cancer stage. The specificities of EBV DNA and IgA-VCA were 98% (96-99%) and 96% (91-98%), respectively. Among 36 healthy family members with false-positive IgA-VCA results, three-fourths had undetectable EBV DNA, whereas the others had increased EBV DNA concentrations that were significantly lower than in NPC patients. CONCLUSIONS: For diagnosis of NPC, EBV DNA identifies almost all false-negative IgA-VCA cases and gives a 99% diagnostic sensitivity when combined with IgA-VCA. In the screening setting, EBV DNA identifies three-fourths of false-positive IgA-VCA cases. The selective application of EBV DNA in an IgA-VCA-based screening protocol could improve screening accuracy with only moderate increases in cost.     

Analysis of Epstein-Barr virus in hodgkin's disease: Experience of a single university hospital in Korea

Hodgkin's disease is known to be associated with Epstein-Barr virus (EBV) infection in Western countries, and viral nucleic acids and proteins have been identified within Reed-Sternberg (RS) cells, which are the histopathologic hallmark of the disease process. Twenty-five cases of Hodgkin's disease from a single university hospital in Korea were studied for evidence of EBV by in situ hybridization for EBV DNA and RNA and immunohistochemistry for an EBV latent protein. EBV nucleic acids were studied by a rapid (60 minutes) in situ hybridization procedure, which utilized biotinylated DNA probes specific for the following nucleic acid sequences: (1) EBV EBER1 RNA (an abundant RNA sequence expressed during latent EBV infection), (2) EBV Notl repeats (a tandemly repeated DNA sequence, which has been established to identify amplified EBV genome in lytic EBV infection), and (3) BAM HI W (a DNA sequence reiterated 11 times within the viral genome). In addition, immunohistochemistry for EBV latent membrane protein, a protein that is capable of inducing cellular transformation in cell culture, was also performed. EBV was identified within the neoplastic RS cells by at least one method in 19/25 cases (76%). The mixed cellularity subtype was the most common subtype associated with EBV infection (11/13–85%). In situ hybridization for EBV EBER1 RNA was the most sensitive method for EBV detection and was present in 17/25 cases. A significant proportion of Korean Hodgkin's disease cases is associated with EBV infection. © 1994 Wiley-Liss, Inc.     

Sequence analysis and variation of EBNA-1 in Epstein-Barr virus-related herpesvirus of cynomolgus monkey

OBJECTIVES: The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is an important protein for immortalization and tumorigenesis of infected cells. EBNA-1 gene variants may play a role in tumorigenesis. We determined the nucleotide and amino acid (aa) sequences of EBNA-1 in EBV-related herpesviruses from cynomolgus monkeys (cynomolgus-EBV) which induced malignant lymphomas in its natural host and in rabbits, and compared them with sequences of EBV and other lymphocryptoviruses (LCVs). METHODS: Polymerase chain reaction and direct sequencing methods were performed using extracted DNA from cynomolgus-EBV-infected cell lines. RESULTS: The amino acid sequences of cynomolgus-EBV EBNA-1 from two cell lines (Si-IIA: 588 aa; Ts-B6: 619 aa) which are antigenically cross-reactive to human EBV EBNA-1 showed homology with human EBV (Si-IIA: 53%; Ts-B6: 58%) and other LCVs from baboons (54 and 52%) and rhesus monkeys (60 and 58%), especially in the C-terminal unique domain. Homology of the EBNA-1 sequence between Si-IIA and Ts-B6 was 92%. The sequence difference between EBV and the related LCVs was manifested mainly in the length of the internal repeat 3-corresponding region, which contains serine in the glycine/alanine repeat region of nonhuman LCVs. CONCLUSION: Sequence variation of cynomolgus-EBV EBNA-1 from different cell lines was observed. However, their sequences show a relatively high homology with human EBV and share the common features of EBNA-1 of EBV and other LCVs.     

Chemiluminescence detection of epstein-barr virus DNA with an oligonucleotide probe

A sensitive and relatively fast chemiluminescence dot blot hybridization assay for Epstein-Barr virus (EBV) DNA was established. A specially designed oligonucleotide probe labeled directly by alkaline phosphatase (AP) and highly sensitive chemiluminescent substrate CDP-star for AP was used in the hybridization assay. About 4 pg purified EBV DNA target can be detected, improved about 10-fold in sensitivity compared with common colorimetric detection. Preliminary EBV clinical testing was carried out on samples of eight cases from patients with nasopharyngeal carcinoma (NPC) (six cases) and nasopharyngeal lymphadenosis (two cases). The detection results in positive rate agreement with control PCR detection indicate that the assay is specific and sensitive for EBV. The whole detection procedure can be finished in 6 h. This method might be useful in EBV clinical diagnosis.     

Low intrathecal immune response of anti-EBNA-1 antibodies and EBV DNA from multiple sclerosis patients

Numerous studies have been carried out to determine whether an Epstein-Barr virus (EBV) infection can be considered a risk factor for multiple sclerosis (MS), following the evidence of an increase in IgG response to nuclear antigen-1 (EBNA-1) in both serum and cerebrospinal fluid (CSF) from MS patients. However, the possible interaction between EBV and MS has still not been well characterized, and the possible pathogenic role is yet to be determined. A case-control study (76 cases and 75 controls) was conducted to investigate anti-EBV antibodies synthesis in serum and CSF through intrathecal specific IgG synthesis to EBNA-1, as well as the presence of EBV DNA in plasma, peripheral blood mononuclear cells, and CSF from MS patients. Intrathecal EBNA-1 specific IgG synthesis was detected in 6.6% MS patients and in 17.3% controls. No EBV DNA was found in plasma or CSF, and our findings showed no evidence of high intrathecal EBNA-1 specific IgG synthesis or of significant EBV DNA in CSF in MS patients.     

江苏省扬州地区胃癌组织中EB病毒感染与p53蛋白表达的相关性

目的探讨Epstein-Barr病毒(EBV)感染与p53蛋白表达异常在扬州地区胃癌发生发展中的作用及其相关性。方法采用原位杂交法检测胃癌石蜡标本中EB病毒编码的小RNA(EBER1),免疫组化检测胃癌组织中p53蛋白的表达情况。结果 96例胃癌组织中有15例呈EBER1阳性,有明显淋巴细胞浸润的11例,均为管状腺癌。EB病毒阳性胃癌(E-BVaGCs)组织中p53蛋白表达显著高于EB病毒阴性胃癌(EBVnGCs)组织。结论 EBV感染与本地区胃癌的发生有一定的相关性,癌间质中大量淋巴细胞浸润可能是EB病毒感染的重要病理学特征。胃癌组织中EBV感染与p53蛋白的异常表达密切相关。