核酸提取方法的研究进展

随着近年来分子生物学技术的高速发展,以核酸杂交、核酸扩增和核酸序列分析为代表的分子诊断和检测技术在诸多领域中日益凸显出至关重要的作用。新型的分子生物学检测技术包括聚合酶链反应（PCR）、微芯片、高通量测序等,然而,所有现代分子生物学检测技术首先面临的问题就是如何从复杂多样的生物样本中迅速有效地分离和提取所需要的基因组核酸,而且抽提后的核酸质量及其完整性都会直接影响到随后的实验结果。目前,全世界的研究者在核酸分离提取的技术方法上也取得了许多突破性的进展,本文将就新型的核酸分离提取方法作一综述。     

核酸提取方法的改进

实时荧光定量逆转录聚合酶链反应（RT-PCR）广泛应用于RNA病毒的核酸检测,而前期的RNA提取效率高低直接影响到检测效率,以往病毒RNA提取主要采用异硫氰酸胍-酚-氯仿及TRIzol法[1-3]等,但操作步骤都较繁琐,且易造成污染而出现假阳性。     

荧光定量PCR检测HCV RNA两种核酸提取法的比较

本文探讨两种核酸提取方法对HCVRNA结果的影响。现报告如下。 1材料和方法 1.1标本来源采集2009年2月～2009年10月徐医附院肝炎门诊疑似丙肝感染患者血清标本92份,男56人,年龄（16～83）岁;女36人,年龄（16～75）岁。     

两种提取红细胞膜蛋白方法的比较

两种提取红细胞膜蛋白方法的比较肖毅杨粉莲（第四军医大学微生物学教研室＊西京医院输血科，西安７１００３２）在制备抗血型抗原单克隆抗体时，为了提高红细胞血型抗原的免疫效果，获得更多的阳性克隆，我们比较了两种提取红细胞膜蛋白的方法。１材料和方法１．１低渗溶...     

几种线粒体DNA提取方法的比较

目的将提取线粒体DNA的碱变性法、Triton法、改进高盐沉淀法加以比较,以得到最方便快速提取线粒体DNA的方法.方法采用健康成年雄性Wistar大鼠,取回肠上皮细胞样本18份,每份含3×10?6细胞,每6份分别用碱变性法、Triton法、改进高盐沉淀法提取线粒体DNA,紫外分光光度法定量.再用琼脂糖凝胶电泳和线粒体ATPase6亚基基因PCR扩增产物鉴定所提取的线粒体DNA.结果3种方法提取线粒体DNA量差别明显改进高盐沉淀法量最多,为(1.26±0.23)μg;碱变性法次之,为(0.52±0.18)μg;Triton法为(0.31±0.16)μg.A260/A280均大于1.7.说明改进高盐沉淀法提取线粒体DNA具有操作简单,产量多的优点.将改进高盐沉淀法提取线粒体DNA用于PCR扩增,测定出了线粒体DNAATPase8亚基基因序列,说明该法所提取mtDNA可用于mtDNA测序.讨论和结论线粒体在细胞凋亡,衰老及程序化死亡中发挥重要作用.已在多种疾病中发现mtDNA突变.对线粒体疾病的分子遗传机理的研究可以进一步阐明这些疾病的病因,并为治疗提供理论指导.胡义德、Tamura和Palva等用碱变性法提取了人血白细胞、心肌等组织中mtDNA;戴纪刚等建立的Triton法先除去细胞核,再分离提取胞浆中mtDNA.而高盐沉淀法通过SDS(十二烷基硫酸钠)破坏细胞膜、核膜,使蛋白质变性,从而游离出核酸,EDTA抑制细胞中DNA酶的活性,蛋白酶K进一步将蛋白质降解成小肽,加入饱和乙酸钠后,绝大部分线性大分子量DNA和蛋白质在SDS作用下变性形成沉淀,环状mtDNA仍为自然状态,通过高速离心,即可得到mtDNA.3种方法各有优缺点碱变性法操作时间较短,要求条件比较严格,不易重复;Triton法通过核质分离提取mtDNA操作时间短,但产量低,易降解.而高盐沉淀法操作简单,易重复,产量多,可依需用量扩大反应体系,使mtDNA质量得以容易控制.     

狂犬病毒糖蛋白基因重组腺病毒与同一抗原核酸疫苗的联合免疫研究

目的 研究联合运用狂犬病毒糖蛋白基因重组腺病毒与同一抗原核酸疫苗对小鼠的免疫效果。方法 构建狂犬病毒糖蛋白（GP）的真核表达质粒pcDNA3.1/CVS-N2c GP作为核酸疫苗，瞬时转染COS－7细胞，间接免疫荧光试验检测pcDNA3.1/CVS-N2c GP的表达；以基因枪方法进行初次免疫接种和首次加强免疫，以表达同一抗原的复制缺陷型重组腺病毒通过鼻腔接种进行第二次加强免疫；ELISA试验检测血清狂犬病毒特异性IgG抗体，快速荧光灶抑制试验（RFFTT）检测狂犬病毒中和抗体。结果 间接免疫荧光试验表明pcDNA3.1/CVS-N2c GP能有效地表达GP于转染的细胞膜上，ELISA试验表明小鼠接受基因核核酸免疫后，仅诱导产生低水平的特异性抗体，而且重组腺病毒进行加强免疫后，特异性抗体水平显著提高。结论 联合运用狂犬病毒糖蛋白基因重组腺病毒与同种抗原核酸疫苗可克服它们单独使用时各自的缺点，能有效地诱导小鼠产生抗狂犬病毒特异免疫。     

两种全血基因组DNA提取法的比较

目的比较经典的酚／氯仿法和人全血基因组DNA小量快速提取试剂盒抽提法两种方法提取人全血基因组DNA的纯度及总量的差异。方法收集静脉血5毫升,共132人份。分别采用上述两种方法提取基因组DNA,用紫外分光光度仪及琼脂糖凝胶电泳检测其纯度和总量,采用统计学t检验,数据以均数±标准差（x±s）表示。结果经典的酚／氯仿法和人全血基因组DNA小量快速提取试剂盒抽提法两种方法提取基因组DNA纯度用0D260／OD280表示分别为：1．65＋0．12,1．86＋0．15。基因组DNA总量分别为28．3＋3．02,33．8＋3．24。结论从提取的基因组DNA纯度和总量来比较,人全血基因组DNA小量快速提取试剂盒抽提法抽提方法较好。     

一种新的DNA提取方法TLS法

从不同组织、细胞或外周血中制备高分子ＤＮＡ，是进行分子水平研究的先决条件。ＤＮＡ以核蛋白形式存在于细胞核中，制备ＤＮＡ的原则是既要将蛋白质、脂质、多糖及小分子杂质分离干净，又要得到高分子量ＤＮＡ。近年来采用蛋白酶Ｋ水解的方法，使这一问题得到较好的解决...     

四种核酸荧光染料在琼脂糖凝胶中染色特性的比较分析

目的比较几种核酸染料(Genefinder、Geneview、SYBER greenI)对于琼脂糖凝胶中DNA/RNA的染色效果并分析其取代溴化乙锭(EB)用于常规电泳后凝胶中核酸染色的可能性。方法分别使用四种核酸染料对凝胶中的RNA/DNA进行染色并对结果进行比较分析。结果四种染料对RNA/DNA染色结果无明显差异(P>0.05)。结论新型核酸染料Genefinder、Geneview和SYBER greenI能够代替EB,用于常规凝胶中的DNA/RNA染色。     

人3型腺病毒荧光定量PCR检测方法的建立

目的通过建立人3型腺病毒荧光定量PCR检测方法,快速鉴定人3型腺病毒感染,为早期诊断提供依据。方法以人3型腺病毒感染的细胞和常见的呼吸道消化道感染的病原体为研究对象,根据病毒六邻体高度保守的序列,设计荧光定量PCR反应体系,并分析实验的敏感性和特异性。结果人3型腺病毒TCID50细胞培养液中病毒核酸最低检出稀释度是10^-6,对应的拷贝数分别是5．802×10^3copies／mL和6．968×10^2copies／mL．呼吸道和消化道感染常见的病原体未出现交叉反应。结论荧光定量PCR的应用,可有效缩短检测时间,提高检测的敏感度。     

Comparisons of Three Automated Systems for Genomic DNA Extraction in a Clinical Diagnostic Laboratory

Purpose

The extraction of nucleic acid is initially a limiting step for successful molecular-based diagnostic workup. This study aims to compare the effectiveness of three automated DNA extraction systems for clinical laboratory use.

Materials and Methods

Venous blood samples from 22 healthy volunteers were analyzed using QIAamp® Blood Mini Kit (Qiagen), MagNA Pure LC Nucleic Acid Isolation Kit I (Roche), and Magtration-Magnazorb DNA common kit-200N (PSS). The concentration of extracted DNAs was measured by NanoDrop ND-1000 (PeqLab). Also, extracted DNAs were confirmed by applying in direct agarose gel electrophoresis and were amplified by polymerase chain reaction (PCR) for human beta-globin gene.

Results

The corrected concentrations of extracted DNAs were 25.42 ± 8.82 ng/µL (13.49-52.85 ng/µL) by QIAamp® Blood Mini Kit (Qiagen), and 22.65 ± 14.49 ng/µL (19.18-93.39 ng/µL) by MagNA Pure LC Nucleic Acid Isolation Kit I, and 22.35 ± 6.47 ng/µL (12.57-35.08 ng/µL) by Magtration-Magnazorb DNA common kit-200N (PSS). No statistically significant difference was noticed among the three commercial kits (p > 0.05). Only the mean value of DNA purity through PSS was slightly lower than others. All the extracted DNAs were successfully identified in direct agarose gel electrophoresis. And all the product of beta-globin gene PCR showed a reproducible pattern of bands.

Conclusion

The effectiveness of the three automated extraction systems is of an equivalent level and good enough to produce reasonable results. Each laboratory could select the automated system according to its clinical and laboratory conditions.     

Detection of adenoviruses in stool specimens by nucleic acid spot hybridization

Nucleic acid hybridization was used for the detection of adenovirus DNA in stool specimens, and the results were compared with those obtained by a radioimmunoassay (RIA) for adenovirus hexon antigen. DNA from 40 specimens, 18 of which were positive by RIA, were spotted onto nitrocellulose filters and analyzed by hybridization using radioactively labeled adenovirus-2 DNA or a cloned DNA fragment from enteric adenovirus-41 as probes. With the adenovirus-2 DNA probe, 15 of the 18 RIA-positive specimens were also positive in the hybridization assay, and one of the RIA negative specimens was also scored as positive. The cloned adenovirus-41 fragment gave a positive signal with five specimens, all of which were also detected with the adenovirus-2 DNA probe. The results show that hybridization is an alternative method for detection of adenovirus in stool specimens. The sensitivity of the assay is comparable to that of the RIA.     

长引物快速PCR结合核酸试纸条法可视化检测四种病原体

目的建立一种基于病原体核酸分子的血液安全性检定方法,可视化区分血液中乙肝病毒（HBV）、丙肝病毒（HCV）、艾滋病毒（HIV）和梅毒螺旋体（TP）。方法利用长引物PCR扩增四种病原体核酸,循环步骤由传统的三步简化为两步。将修饰有小分子抗原的扩增产物点样于固定有相应抗体的纳米颗粒试纸条上,肉眼判读显色结果。优化了长引物PCR对于四种病原体扩增的退火和变性温度,并评价了该方法用于病原体阳参及血清样本的检测准确性。结果四种病原体核酸扩增的退火温度分别为76℃、78℃、76℃和78℃;变性温度为84℃、90℃、84℃和88℃;本方法能成功区分四种病原体的阴阳性样本,准确性高;扩增时间仅为40 min/40个循环,检测为10 min。结论该方法能够用于四种病原体核酸的可视化检测,与传统的检测方法相比,检测速度快,成本低,无需昂贵的仪器,易于广大基层医院普及。     

粪便不同保存方法对动物基因组DNA提取效果的影响

目的为了探索一种既能使野外采集的粪便样品DNA保存完好,又方便带回实验室用于分子水平研究的粪便保存方法。方法本研究通过将普氏原羚的粪便在野外分别采取60℃干燥后低温保存,75％乙醇保存,100％乙醇保存,直接低温(保温箱中加生物冰袋)保存,在1个月内、5个月后、8个月后对粪便DNA进行抽提,并将提取的DNA作为模板进行PCR扩增,基因克隆测序。结果短期内均可得到高质量的DNA,DNA大小在23kb左右,电泳带型整齐,无降解,线粒体的扩增结果也完全一致,而用100％乙醇保存粪便DNA的时间更为长久。结论不同的保存方法提取动物基因组效果不一样。     

Quantitation of Hepatitis B Virus Genomic DNA by Real-Time Detection PCR

Quantitation of hepatitis B virus (HBV) DNA in serum is a useful method for the monitoring of HBV replication. We attempted to develop a quantitative assay system for HBV DNA that is more sensitive, accurate, and reproducible than existing systems. We detected HBV DNA by real-time detection PCR (RTD-PCR) based on Taq Man chemistry. The efficacy of this assay was evaluated by quantitatively measuring sequential levels of synthetic DNA and DNA in clinical serum samples. The detection limit of this system was as few as 10 DNA copies/reaction. A linear standard curve was obtained between 10(1) and 10(8) DNA copies/reaction. The coefficient of variation for both intra- and interexperimental variability indicated remarkable reproducibility. This system detected HBV DNA in 100% of chronic hepatitis B patients tested and never detected HBV DNA in healthy volunteers who were negative for HBV markers. These observations suggest that RTD-PCR is an excellent candidate for a standard HBV quantification method.     

Comparison of Methods for the Extraction of DNA from Formalin-Fixed,Paraffin-Embedded Archival Tissues

Aim: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues.Methods: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability.Results: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification.Conclusion: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE.     

Comparison of Five PCR Methods for Detection of Helicobacter pylori DNA in Gastric Tissues

Five different PCR methods for the detection of Helicobacter pylori were evaluated. The results of this study indicate that of the five PCR methods examined, the ureC (glmM) gene PCR is the most sensitive and specific for the detection of H. pylori in gastric biopsy specimens.     

Improving Molecular Detection of Fungal DNA in Formalin-Fixed Paraffin-Embedded Tissues: Comparison of Five Tissue DNA Extraction Methods Using Panfungal PCR

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative microorganisms in formalin-fixed paraffin-embedded (FFPE) tissue is essential (20). Tissue samples collected and processed for pathological diagnosis represent a unique source of archived and morphologically defined disease-specific biological material (24). Histopathologic examination remains one of the major diagnostic tools in mycology because it permits rapid, presumptive identification of fungal infections. In recent years, however, there have been cases with discrepant histologic and culture results at final diagnosis; such discrepancies could lead to unnecessary pharmaceutical exposure and/or inappropriate treatment (17, 24).Recent efforts to improve the sensitivity and specificity of diagnostic tests have focused on culture-independent methods, in particular, nucleic acid-based methods, such as PCR assays. PCR-based detection of fungal DNA sequences can be rapid, sensitive, and specific and can be applied to fresh and FFPE tissues (16). The majority of fungal assays target multicopy loci, in particular, the ribosomal DNA (rDNA) genes (18S, 28S, and 5.8S) and the intervening internal transcribed spacer (ITS) regions (ITS1 and ITS2) in order to maximize the yield of amplified DNA and allow high specificity (9).Several protocols have been described for the extraction of DNA from fresh tissue, blood, and cells in cultures, but extraction from FFPE tissues is difficult because the material is frequently scarce and degraded and often contains remnants of either substances that inhibit the amplification reaction or chemicals, such as formalin, that inhibit the proteinase K used in the DNA extraction procedure. In general, FFPE tissue requires special protocols in order to extract small amounts of DNA suitable for amplification (6, 7, 10, 18).In this work, we evaluated five commercial kits for the extraction of high-quality DNA from FFPE tissues that can be applied in molecular studies. To the best of our knowledge, three of the five protocols have not been previously evaluated in the context of extracting fungal DNA. After DNA extraction, the subsequent molecular analyses included two housekeeping gene PCR assays and three different panfungal PCR assays, followed by sequencing of the DNA fragments obtained. The protocols were assessed for time spent in performing the procedure, quality of DNA detection, and efficiency of fungal-DNA detection.