DNA甲基化检测助力肿瘤早期筛查和诊断

DNA甲基化是国内外癌症早筛采用的常用检测指标。在致癌因素的作用下,抑癌基因启动子区的高甲基化可导致基因表达下调或关闭,从而激活原癌基因的表达,促使肿瘤发生。用于DNA甲基化检测的样本主要采用脱落细胞、血液样本和石蜡包埋组织。常用的检测方法包括特异性聚合酶链反应（methylation-specific polymerase chain reaction, MSP）、核酸质谱、甲基化芯片、亚硫酸氢盐测序、第二代测序技术等。与突变检测相比,DNA甲基化的检测优势在于其肿瘤特异性高、可检测位点多、信号丰度高,且可实现组织溯源。目前,DNA甲基化检测被主要运用在临床脑胶质瘤用药指导、肺癌辅助诊断和高危分流、膀胱癌高危分流和复发监测、宫颈癌高危分流、结直肠癌和胃癌早期筛查及复发监测。在实现大规模临床常规应用前,需注意精准定位DNA甲基化检测的应用场景,相对于耐受性良好的内镜检查,其高危分流的刚需性可能会下降。在辅助诊断病理诊断灵敏度较低的肿瘤、肿瘤监测和预后评估方面,DNA甲基化检测有着重要的作用。随着检测流程和质量管理的规范,DNA甲基化检测会被更广泛地运用于临床,提高肿瘤的早筛和诊断灵敏度...     

DNA甲基化分析技术的研究进展

近年来,在肿瘤表观遗传学研究领域,DNA甲基化受到越来越多的关注.原因可能是DNA的分子结构相对稳定,提取及保存更为容易,在不同的组织标本中均能被有效检出.然而,要将其真正应用于临床,还亟需发展一种兼具灵敏性、特异性及可操作性的检测手段.目前一般认为有两种甲基化检测思路[1]:即甲基化分型和甲基化组学.前者通常是对单个基因或较少基因进行检测,后者则是对全基因组进行甲基化分析,又叫甲基化谱.本文围绕这两类检测技术及其在临床检验方面的应用价值进行综述. 1 甲基化分型 1.1 甲基化特异的聚合酶链反应(MSP) MSP的原理是将重亚硫酸盐处理后的DNA作为模板,在基因启动子5’端富含胞嘧啶鸟嘌呤二核苷酸(CpG)位点的甲基化多发区域设计两对引物,一对针对甲基化的模板,另一对针对未甲基化的模板,通过凝胶电泳法,判定哪一对引物能有效扩增出目标基因,以此作为甲基化与否的标准.此法简单且易于操作,是目前应用最为广泛的甲基化定性分析手段.     

肿瘤细胞分子标志物-启动子甲基化

近年来，在许多癌症相关基因中都发现了启动子区域甲基化状态改变的现象，使得DNA甲基化这一后生性调控作用的重要性被重新认识。肿瘤是遗传因素和后天因素共同作用的多因子疾病。本文总结了DNA启动子甲基化与肿瘤关系的研究现状，阐述了启动子甲基化作为肿瘤诊断指标的可能性，并对甲基化各种分析方法的优缺点作了比较，最后介绍了MSP方法的原理，特点，实验中的问题及解决方法。     

DNA甲基化检测方法的进展

DNA甲基化是重要的表观遗传学改变之一,肿瘤细胞中DNA甲基化形式包括基因组的整体水平低甲基化及特定基因的高甲基化.尤其是抑癌基因启动子区的高甲基化,可直接阻碍转录因子与启动子结合,从而使抑癌基因不能转录或转录水平降低.因此,基因启动子的异常甲基化可作为候选的肿瘤标志物.大量研究通过对肿瘤患者血清及其他体液中的凋亡肿瘤细胞的DNA甲基化检测辅助诊断肿瘤及判断预后,包括乳腺癌患者的乳头抽吸物,肺痛患者痰及支气管灌洗液,前列腺癌患者的尿液及其他多种肿瘤患者的血浆及血清[1].     

基于血液DNA甲基化在肝癌诊断中的研究进展

肝癌是世界范围内致死率最高的癌症之一,早期诊断率低,通常在发现肝癌时患者已经处于晚期。早期诊断是降低肝癌患者死亡率的重要手段,但目前尚无诊断肝癌的可靠血液学分子标志物。循环游离DNA甲基化检测是诊断肿瘤的一种潜在的非侵入式方法。本文结合循环游离DNA甲基化在肝癌诊断中的最新研究,主要综述血液中单个DNA甲基化标志物、多个DNA甲基化标志物联合及DNA甲基化标志物联合甲胎蛋白(AFP)在肝癌诊断方面的研究进展。     

不同甲基化检测技术对模拟肺癌患者血浆中APC基因甲基化的检测

目的 研究模拟肺癌患者血浆DNA中APC基因甲基化最低检测限,确定不同甲基化检测技术在早期肺癌诊断中的价值.方法 用普通甲基化特异性基因扩增（methylation specific PCR,MSP）方法检测肺癌细胞株NCI-H460细胞APC基因,以酚-氯仿经典方法提取NCI-H460细胞DNA,紫外分光光度计定量并以10倍稀释的浓度梯度依次投入相同的健康人血浆200μl中去,得到模拟肺癌患者血浆,利用磁珠核酸提取方法从模拟血浆样本中提取血浆DNA.对血浆DNA模板进行亚硫酸氢盐化学修饰,并以Sybr-Green Ⅰ为染料进行实时荧光定量MSP检测,同时进行普通MSP检测.结果 NCI-H460细胞中的APC基因启动子1A区719位点存在甲基化,实时荧光定量MSP检测模拟肺癌患者血浆APC基因甲基化的最低检测限为在200 μl血浆中能检测出10^2个肿瘤细胞的DNA,而以普通MSP检测,200 μl血浆中需投入10^3个以上肿瘤细胞的DNA才有弱阳性条带.实时荧光定量MSP检测技术比普通MSP检测的敏感性至少高出10倍.结论 实时荧光定量MSP比普通MSP检测灵敏度高,该技术用于肺癌患者血浆DNA甲基化检测,可能有助于肺癌早期诊断.     

DNA甲基化及其在肿瘤分子诊断中的前景

DNA甲基化是研究最为深入的表观遗传学机制。该机制的异化可导致基因表达的异常及基因组稳定性的降低，继而促进肿瘤发生和发展。启动子CpG岛的高甲基化已被认为是与遗传性缺陷同等重要的、造成抑癌基因在肿瘤中失活的分子生物学机制。虽然有关DNA甲基化肿瘤特异性异化谱式信息仍极有限，但其在肿瘤临床诊断和预后中的巨大潜力已开始得到重视。现结合我们近3年的工作和该领域的最新进展，对DNA甲基化作为肿瘤临床诊断标志物的应用前景进行评估。     

循环游离DNA甲基化研究进展

循环游离DNA是存在于人体外周循环中的游离DNA。循环游离DNA甲基化与多种恶性肿瘤相关,其作为分子标志物有自身特性和优势。本文就循环游离DNA的来源和生物学特性、甲基化改变及其作为相关肿瘤标志物、检测方法等研究进展作一综述。     

SYBR Green Ⅰ荧光定量PCR检测DNA甲基化方法的建立

近期，表观遗传学与肿瘤发生关系的研究日益增多。DNA甲基化是表观遗传学的重要组成部分，并已成为肿瘤早期诊断的研究热点。甲基化特异性PCR（MSP）是检测DNA甲基化的常用方法之一，该方法灵敏度和特异度高，但操作繁琐，难以自动化。TaqMan荧光定量PCR法，即Methylight在MSP的基础上设计1条TaqMan探针，该方法的准确性和检测的灵敏度都很高。然而，因需要设计合成特异的探针而使其的应用受限，而且TaqMan探针昂贵。本研究建立SYBRGreenⅠ荧光定量PCR（FQ—PCR）检测DNA甲基化的方法，通过临床标本检测和对比试验，以证明其与Methylight的检测效能，以便为DNA甲基化检测的临床应用提供工具。     

p16基因甲基化对胃癌的早期诊断

目的:检测胃癌患者癌组织及全血DNAp16基因甲基化,并探讨其在胃癌早期诊断中的作用。方法:收集新鲜的胃癌组织及血液标本4 4例,胃溃疡等胃部良性疾病患者标本2 0例。采用甲基特异性PCR(MSP)技术分别检测肿瘤组织DNA和全血DNA中p16基因启动子甲基化。结果:38.6 % (17/ 4 4 )胃癌组织出现p16基因甲基化,出现甲基化组织对应的血液标本中88.2 % (15 / 17)出现p16基因甲基化。胃溃疡等胃部良性疾病患者标本中未发现甲基化。肿瘤组织中p16基因的甲基化状况与全血中出现p16基因甲基化关系密切(P<0 .0 1)。结论:胃癌组织及血液标本中p16基因甲基化的检测有助于胃癌的早期诊断。     

Principle and applications of digital PCR

Digital PCR represents an example of the power of PCR and provides unprecedented opportunities for molecular genetic analysis in cancer. The technique is to amplify a single DNA template from minimally diluted samples, therefore generating amplicons that are exclusively derived from one template and can be detected with different fluorophores or sequencing to discriminate different alleles (e.g., wild type vs. mutant or paternal vs. maternal alleles). Thus, digital PCR transforms the exponential, analog signals obtained from conventional PCR to linear, digital signals, allowing statistical analysis of the PCR product. Digital PCR has been applied in quantification of mutant alleles and detection of allelic imbalance in clinical specimens, providing a promising molecular diagnostic tool for cancer detection. The scope of this article is to review the principles of digital PCR and its practical applications in cancer research and in the molecular diagnosis of cancer.     

裂解液加热煮沸法在血清HBV DNA抽提中的应用

目的建立裂解液加热煮沸法抽提血液HBV DNA的方法。方法选取浓度分别为10^6、10^5、10^4、10^3copies/ml的HBV DNA阳性血清,采用5种方法抽提HBV DNA,并用SYBR Green Ⅰ荧光定量PCR检测抽提产物。结果在HBV DNA浓度为10^6、10^5、10^4copies/ml时5种抽提法（异硫氰酸胍加热煮沸/盐酸胍加热煮沸/kit/SDS加热煮沸/chelex-100加热煮沸）均能得到阳性结果,而在HBV DNA为低浓度10^3copies/ml时,只有异硫氰酸胍加热煮沸/盐酸胍加热煮沸/chelex-100加热煮沸能得到阳性结果。结论chelex-100裂解液加热煮沸抽提HBV DNA操作简便、省时、经济,为血液标本HBV基因检测在临床上的大规模应用奠定基础。     

高通量人乳头瘤病毒分型基因诊断方法的研究

目的 建立一种可靠、简便经济、快速、高通量的人乳头瘤病毒(HPV)分型检测新方法，并推向临床应用。方法 以各型HPV通用引物行聚合酶链反应(PCR)扩增127例患者的尖锐湿疣组织和宫颈刮片，将荧光偏振检测技术与模板指导的末端延伸反应(TDI—FP)结合，应用探针杂交延伸反应对临床样本扩增产物进行HPV分型：HPV6，11，16，18，31，33，35，58检测，并以DNA序列测定结果为参照。结果 在78例患者的尖锐湿疣组织块中HPV检出率100％，其中两型混合感染14例，占18％，三型混合感染5例，占7％，三型以上混合感染1例。49例患者的宫颈刮片HPV检出率为77％，其中两型混合感染6例，占感染者的17％，三型混合感染3例，占感染者的8％，三型以上混合感染1例。但DNA序列测定方法仅能检出单独一型感染，未检出多重混合感染，两法对单一型检出符合率100％。结论 本研究初步建立了通用、特异、便捷，无需标记探针，可自动化高通量用于临床的人乳头瘤病春分型基因诊断方法。     

ME-PCR for the identification of mutated K-ras in serum and bile of pancreatic cancer patients: an unsatisfactory technique for clinical applications

Our aim was to assess the clinical reliability of mutated K-ras detection in serum or bile for the diagnosis of pancreatic cancer using ME-PCR. DNA was extracted from 1 ml serum obtained from 29 patients with pancreatic cancer and 12 control subjects. ME-PCR was optimized using a mixture of normal DNA added with different amounts of mutated DNA. The analysis of sera obtained from the 29 patients and of bile obtained from 11 pancreatic cancer patients demonstrated the presence of mutated K-ras in two (6.9%) and four cases (36%). By contrast K-ras was not amplifiable in any of the 12 serum samples obtained from healthy controls. In conclusion the DNA obtained from pancreatic cancer patients' sera is suitable for K-ras amplification and for the identification of codon 12 point mutations. However ME-PCR alone has an unsatisfactory sensitivity for the detection of pancreatic cancer using serum DNA as starting template.     

提高SAF基因启动子区DNA模板聚合酶链式反应的扩增效率

背景：血清淀粉样蛋白A转录激活因子基因启动子区鸟嘌呤和胞嘧啶的含量偏高,常规聚合酶链式反应扩增不易成功。目的：探索提高血清淀粉样蛋白A转录激活因子基因启动子区富含鸟嘌呤和胞嘧啶的DNA模板聚合酶链式反应扩增方案及优化的扩增体系。方法：提取THP-1细胞基因组DNA作为模板,通过97℃高温预变性及最佳退火温度的探索优化聚合酶链式反应的反应条件,在聚合酶链式反应扩增体系中添加不同浓度的增强剂二甲基亚砜改善反应体系。建立提高血清淀粉样蛋白A转录激活因子基因富含鸟嘌呤和胞嘧啶的启动子区聚合酶链式反应扩增效率的方法,同时评估不同浓度二甲基亚砜对聚合酶链式反应扩增结果的影响。结果与结论：97℃高温预变性使模板DNA充分解链,同时提高退火温度至60℃,有利于提高聚合酶链式反应扩增产率;反应体系中添加2％二甲基亚砜可以提高血清淀粉样蛋白A转录激活因子基因启动子区聚合酶链式反应产物的特异性和产率,但是鸟嘌呤和胞嘧啶含量不同对增强剂的依赖不一样。结果证实,高温预变性和较高的退火温度可以保证充分解链的模板DNA与引物特异性结合,但是DNA模板鸟嘌呤和胞嘧啶含量的不同对增强剂二甲基亚砜的依赖有差异。     

Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction

For the detection of food born bacteria by polymerase chain reaction (PCR) in food products, an internal amplification control (IAC) is required in order to prevent false negative results that might be caused by PCR inhibitors. In the present study, two IACs were constructed using two different methods. These IACs were designed in a way that the same primer pair can be used to amplify the target DNA and coamplify the IAC. The first IAC with a size of approximately 200 bp was constructed by deleting a part of the amplicon of the original target DNA (500 bp) between the two primer sites to produce an IAC smaller than the target DNA. The second IAC with a size of approximately 600 bp was synthesized in a one step PCR reaction. The primers used in this reaction possessed 5' over-hanging ends, which were identical to the primers used in the diagnostic reaction, whereas their 3' ends were complementary to the (pUC19) predetermined DNA sequence of defined length and sequence. The concentration of IACs appeared to be critical. Too much IAC DNA template would out-compete the target DNA template, thus giving a false negative result. However the use of an optimal IAC concentration increased the reliability of the PCR assays and appeared to be useful for food diagnostics.     

A novel application of melting curves: utility of peak area calculation for relative methylation quantification.

BACKGROUND: DNA methylation markers appear to be useful in cancer detection, in evaluating the prognosis associated with disease progression, and in detecting the metastatic potential of tumors. METHODS: We present an approach for relative quantification of methylated gene sequences. The technique combines conventional PCR and LightCycler fluorescence PCR. In the conventional PCR step, bisulfite-modified molecules are selectively enriched, irrespective of their methylation status, and used as template in the second step, the LightCycler fluorescence PCR, which includes methylation-specific primers and fluorescently labeled probes. After amplification, a stepwise increase in temperature leads to the generation of melting curves reflecting fluorescence emission induced by disassociation of the probe-target complexes. Quantification is based on calculation of the peak areas for melting curves using a Gaussian fit curve. The area under this curve indicates the relative amounts of methylated sequences in each sample. RESULTS: By normalizing the peak areas by template concentrations, a methylation index was calculated for each sample. This approach was then used to carry out a relative quantification of p16INK4A gene methylation in bone marrow and blood mononuclear cells from patients with positive translocation status. CONCLUSIONS: If validated in further studies, this approach represents a highly specific technique that can be used in analyzing paired samples of cancer patients.     

Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples

The application of PCR for the direct and sensitive detection of food-borne pathogens is largely affected by the quality of the template DNA prepared from food samples. In the present study, a chemical extraction method of bacterial DNA from spiked milk samples for the direct detection of Staphylococcus aureus and Yersinia enterocolitica was evaluated by PCR. Gene specific primers were designed to target the nuclease (nuc) and the attachment invasion locus (ail) genes of S. aureus and Y. enterocolitica, respectively and used in PCR. A combination of organic solvents, detergents and alkali in the DNA extraction method permitted a detection limit of 10 cfu ml(-1) milk for both the pathogens. When equal numbers of S. aureus and Y. enterocolitica were spiked in milk samples, the individual detection limit was determined to be 10(3) cfu ml(-1) milk. Simultaneous amplification of 482 and 359 bp fragments of the nuc and ail genes was obtained using the primer pairs in a single reaction. Multiplex PCR enabled the detection of 10(4) cfu ml(-1) milk of S. aureus and Y. enterocolitica without any pre-enrichment step. A combination of conventional isolation technique and PCR using DNA extracted by the proposed method was used to test raw milk samples for possible contamination with S. aureus and Y. enterocolitica. The presence of S. aureus in the tested samples was indicated by both the methods while Y. enterocolitica could not be detected in any of the samples. The template DNA extraction method developed in this study is rapid, sensitive and avoids interference from potential PCR inhibitors and demonstrates the potential of detecting multiple pathogens in milk samples without any enrichment.     

Application of the PCR technique for a rapid,specific and sensitive detection of Salmonella spp. in foods

We report here the use of a PCR based assay modified by us for the detection of Salmonella spp. in foods, based on amplification of a 236 bp Salmonella specific hin/H2 region [Way et al. (1993) Applied and Environmental Microbiology 59, 1473-1479], using Ampli Taq Gold polymerase. Using this assay we were able to detect all the Salmonella serovars tested. The limit of detection was 1 fg of purified target DNA or N x 10(0) (1-3 cells) cfu ml(-1) of pure bacterial culture. This assay could detect N x 10(0) cfu Salmonella cells g(-1) of the food sample unambiguously in presence of endogenous microflora following 6 h enrichment, thus requires a duration of approximately 10 h for the full processing from DNA template preparation, PCR and visualization of DNA product on agarose gel. The main advantage of this PCR detection method is its sensitivity, and specificity. We also tried to adopt DNA template isolation method simply by boiling the bacterial cells thereby reducing the possibility of contamination, cutting the processing time and cost considerably. This can be an added advantage for the use of this system in simple lab setups.     

Enhancing allele-specific PCR for specifically detecting short deletion and insertion DNA mutations

Allele-specific PCR (AS-PCR) has been widely used for the detection of single nucleotide polymorphism. But there are some challenges in using AS-PCR for specifically detecting DNA variations with short deletions or insertions. The challenges are associated with designing selective allele-specific primers as well as the specificity of AS-PCR in distinguishing some types of single base-pair mismatches. In order to address such problems and enhance the applicability of AS-PCR, a general primer design method was developed to create a multiple base-pair mismatch between the primer 3′-terminus and the template DNA. This approach can destabilize the primer–template complex more efficiently than does a single base-pair mismatch, and can dramatically increase the specificity of AS-PCR. As a proof-of-principle demonstration, the method of primer design was applied in colony PCR for identifying plasmid DNA deletion or insertion mutants after site-directed mutagenesis. As anticipated, multiple base-pair mismatches achieved much more specific PCR amplification than single base-pair mismatches. Therefore, with the proposed primer design method, the detection of short nucleotide deletion and insertion mutations becomes simple, accurate and more reliable.