突变敏感性分子开关检测线粒体DNA A1555G位点的条件优化

目的对高保真酶介导的突变敏感性分子开关技术检测线粒体DNA(mt DNA)A1555G位点条件进行优化。方法利用3'硫代磷酸化修饰的突变型引物和野生型引物作为下游引物,在其上游设计一条公共引物分别构成突变引物对和野生引物对,以构建好的包含mt DNA A1555G位点的突变型质粒和野生型质粒为模板,进行高保真聚合酶介导的双向引物延伸反应,对PCR体系中的退火温度、引物浓度、模板浓度等条件优化,通过凝胶成像系统对其PCR结果进行分析确定最佳反应条件。结果分子开关技术检测mt DNA A1555G位点的最佳PCR条件:退火温度为61.0℃,引物浓度为0.6μmol/L,检测模板浓度为103~106copies/μL。结论确定了分子开关技术检测mt DNA A1555G位点的最佳反应条件,为该技术在线粒体DNA的突变筛查中的应用提供依据。     

高分辨率熔解曲线法检测CBS基因SNP的技术优化

目的：探索高分辨率熔解曲线法（highresolutionmelting,HRM）检测胱硫醚β-合酶（CBS）基因SNP位点所需的最佳反应体系和条件,建立快速高效的对CBS基因分型的方法。方法通过普通PCR初步确立引物的退火温度范围,在实时荧光定量PCR（qPCR）-HRM反应中调整确定最佳退火温度。对影响qPCR-HRM的因素包括反应程序、模板DNA、MgCl2再分别进行优化,确立最佳反应体系和反应条件,对在此基础上得出的基因分型结果通过测序验证其准确性,从而建立系统的HRM检测CBS基因SNP的方法。结果HRM检测CBS基因该片段最佳退火温度为62℃,qPCR-HRM最佳反应体系为20μl,引物浓度为0．2μmol/L,Mg2＋为2．5μmol/L,模板DNA为60ng。在优化的体系条件下筛选出的突变型样本经测序验证与实验结果一致。结论HRM可以作为检测SNP准确、经济、高效的方法。     

IgH重排的实时定量PCR检测及反应参数研究

为了探讨以通用引物应用PCR技术扩增克隆性免疫球蛋白重链基因（IgH）重排的最佳实验条件及SYBR Green Ⅰ实时定量PCR检测该基因的可行性，以通用引物对克隆性IgH重排进行扩增，对影响PCR扩增的退火温度、引物浓度、Taq酶用量、dNTP浓度、镁离子浓度、循环次数等实验因素进行了系统研究，找出最佳反应参数。并以通用引物进行了SYBR greenⅠ实时定量PCR对IgH重排基因的检测，测定了该法检测IgH重排基因的敏感性。结果表明：最佳退火温度为60℃，最佳引物浓度为0．8μmol／L，0．5U的Taq酶量效果满意，最适dNTP浓度为100μmol／L，最适镁离子浓度为3．0mmol／L，最佳循环次数为40次。SYBR GreenⅠ实时定量PCR可以实现对克隆性IgH重排的扩增和荧光信号的检测分析，其对IgH重排基因检测的敏感性为10^4／ml。结论：确定了应用PCR技术检测克隆性IgH重排的最适反应条件，实现了用通用引物对克隆性IgH重排稳定、特异的扩增，初步实现了以通用引物和应用SYBR Green Ⅰ实时定量PCR对IgH重排的检测。     

用不均—PCR直接扩增未知基因及基因片段

本文介绍一种改进了循环条件的不均—PCR法。可直接从总DNA而不是从DNA文库中扩增特异的未知片段，它的要点在于，在连续的PCR循环中用两种不同的退火温度有效地扩增了目的片段，同时抑制了非特异产物。合适条件下，一两天内即可获得约4kb的DNA片段。不均一PCR需要两类引物。一类为任意引物，它可以与模板DNA上的许多位点结合，实验发现IO个碱基长度的引物最适合，其最佳参数修正如下：G＋C为60％；终浓度为O．05pNI；反应时高退火温度为55C，低退火温度为42C。另一类引物为特异性引物，它是根据已知的基因组IJNA或CDNA的部…     

假丝酵母SSR-PCR反应体系的优化

目的建立假丝酵母SSR-PCR反应体系,确立最佳反应条件,为将该技术用于临床假丝酵母感染的快速鉴定奠定基础。方法根据引物Tm值设立退火温度梯度,确定适宜的退火温度。利用正交实验,对影响SSR-PCR反应体系的Taq DNA聚合酶、模板DNA、dNTP、引物等4种主要因素,进行优化。结果引物最适退火温度为51℃。假丝酵母最佳的SSR-PCR反应体系为：在25μl的PCR反应体系中加入Taq DNA聚合酶1.0 U、模板DNA为25 ng、dNTP为0.15 mmol.L-1、引物为0.5μmol.L-1。结论采用正交实验优化的SSR-PCR反应体系,操作简便,电泳条带清晰,多态性好,重复性强,可为进一步建立临床假丝酵母感染的快速鉴定提供借鉴。     

KRAS基因突变高分辨率熔解曲线检测技术参数的优化

目的建立KRAS基因突变高分辨率熔解曲线分析法(high resolution melting,HRM)检测方法,确立最佳反应体系和反应条件,为将此技术用于临床基因突变的快速检测奠定基础。方法根据引物Tm值设立退火温度梯度,确定适宜的退火温度。利用正交实验,对影响实时荧光定量PCR(qPCR)-HRM反应体系的模板DNA、Mg2+、引物等3种主要因素进行优化。通过对SW480和MDA-MB-231细胞基因组DNA进行HRM分析检测突变,并直接测序验证,观察反应体系和反应条件的可行性。结果引物最适退火温度为60.8℃。KRAS基因突变qPCR-HRM最佳反应体系为:20μl qPCR-HRM反应体系中,引物浓度为0.5μmol/L,Mg2+为2.5mmol/L,模板DNA为40ng。优化的qPCR-HRM反应体系和反应条件能检测基因突变,和直接测序结果一致。结论采用正交实验优化的qPCR-HRM反应体系,操作简便,电泳条带清晰,熔解曲线峰型单一,特异性好,结果准确可靠,可为进一步建立HRM技术检测基因突变的临床应用提供借鉴。     

病原真菌DNA提取方法及简单重复序列聚合酶链反应体系的优化

背景:真菌DNA的提取在真菌基因工程以及分子生物学研究中占有重要地位.DNA的提取效率,尤其是DNA的质量严重影响实验结果.目的:建立提取病原真菌基因组DNA的方法,探讨简单重复序列-PCR反应体系中各成分的最佳组合.设计、时间及地点:DNA提取方法对照分析及正交实验,于2004-06,2006-12在吉林大学白求恩医学院病原生物学教研室真菌学研究室进行.材料:将接种临床标本的马铃薯葡萄糖液体培养基、马铃薯葡萄糖琼脂培养基、酵母蛋白胨液体培养基28℃培养3-7 d后,选取可疑菌落进行分离、纯化.方法:比较玻璃珠-盐析法、CTAB法、GeneTLE~(TM)抽提法3种不同DNA提取方法对菌体DNA质量的影响;利用正交设计,选择Taq DNA聚合酶、模板DNA、dNTP、引物4个因素,在3个水平上根据L9(3~4)正交表进行试验,对真菌简单重复序列-PCR(SSR-PCR)体系进行优化分析;并通过PCR选择适宜的退火温度及循环次数.主要观察指标:根据扩增获得带型的多态性和特异性,确定最佳反应体系.结果:Gene TLE~(TM)抽提法全部在相应的PCR体系中扩增出目的片段,且带型清楚度、明亮度优于其余两种方法.SSR-PCR反应体系正交试验结果显示,根据尺值大小确定因素显著性顺序为模板DNA(2.67)>Taq DNA聚合酶(2.00)>dNTP(0.67)>引物(0.33);根据ki值大小确定各因素优化水平组合为:模板DNA 30 mg/L,Taq DNA聚合酶1 U,dNTP150 μmol/L,引物0.5 μmol/L.但由于引物作为影响因素的R值小,是非显著因素,因此引物取A水平即0.25 μmol/L.反应条件以53℃退火温度、35个循环次数为最佳.结论:Gene TLE~(TM)提取法提取DNA的效率更高、操作步骤快速简单.根据正交试验的优化结果,SSR-PCR最佳反应体系为模板DNA 30 mg/L,Taq DNA聚合酶1 U,dNTP 150 μmol/L,引物0.25 μmol/L.反应条件以53℃退火温度、35个循环次数为最佳.     

SYBR Green Ⅰ实时定量PCR检测生存素基因的方法建立及条件优化

目的建立SYBRGreenⅠ染料实时定量PCR检测生存素（Survivin）基因定量的方法。方法根据PCR产物荧光强度、循环阈值（Ct值）、标准曲线斜率、相关系数和熔解曲线优化反应体系中各组分的量及反应条件，对引物二聚体消除策略和Ct值获取方式进行评价。并用该方法检测43份胃癌组织Survivin基因扩增情况。结果SYBRGreenⅠ实时荧光定量PCR体系扩增Survivin的最佳组成和条件是：Taq酶2．5U／100μl、MsCl2 2mmol／L、引物浓度0．2μmol／L和退火温度58℃；在PCR循环延伸结束后设置1个低于特异产物退火温度值2℃的荧光读取温度，能有效消除引物二聚体对定量检测的影响；以二次倒数最大值方式确定Ct值，能避免主观因素所致误差。研究建立的SYBRGreenⅠ染料实时定量PCR检测Survivin基因含量方法的灵敏度为10拷贝／μl，线性范围10^1～10^4拷贝μl（r=0．9997），批内变异系数（CV）1．13％-1．91％，批间CV3．31％-4．50％；胃癌组织中Sttrvivin基因扩增率为13．9％（6／43）。结论优化后的SYBRGreenI实时定量PCR方法具有方便、经济、灵敏度高和重复好等特性，可用于Survivin基因含量分析。     

扩增bFGF的条件优化及其真核载体构建

目的 :优化采用PCR技术从PBR3 2 2 bFGF质粒扩增bFGF基因片段的条件并构bFGF真核表达载体 ,为研究bFGF基因转移在骨组织工程学中的应用提供研究基础。方法 :(1)在不同的模板量、Mg2 浓度、退火温度下经PCR扩增bFGF基因 ,琼脂糖凝胶电泳检测。 (2 )载体和PCR产物经EcoRI和HindⅢ酶切、纯化 ,T4连接酶连接 ,转化大肠杆菌 ,抗生素筛选重组质粒。结果 :(1)得到三个最佳参数为 :模板量为 2 μl(5 0 4μg/ml) ;Mg2 浓度为2 0mmol/L ;退火温度为 5 5℃。 (2 )酶切、PCR和DNA序列鉴定均证实插入片段的正确性。结论 :在此优化条件下经PCR反应得到了特异、高效和忠实的bFGF基因片段 ,并成功构建了bFGF真核表达载体。     

纳米金辅助不对称PCR扩增幽门螺杆菌23S rRNA基因的优化和建立

目的:建立纳米金辅助不对称PCR方法扩增幽门螺杆菌23S rRNA基因,为建立基因芯片法检测幽门螺杆菌耐药基因提供试验平台.方法:将幽门螺杆菌23S rRNA基因转化至感受态大肠杆菌JM109,小量抽提阳性构建质粒DNA,以质粒DNA为模板,设计引物对23S rRNA基因进行不对称PCR扩增.对PCR条件中引物浓度及浓度比例、纳米金浓度等进行优化.结果:限制性引物与非限制性引物浓度比例为1 ∶ 60,限制性引物与非限制性引物浓度分别为0.04 pmol/L、2.4 pmol/L时可获得理想的单链和双链DNA,纳米金浓度为0.8 nmol/L时非特异性产物最少,PCR的扩增效率最高.结论:通过对PCR扩增条件的优化,确立了扩增幽门螺杆菌23S rRNA基因纳米金辅助不对称PCR方法的最佳条件,为建立基因芯片法检测幽门螺杆菌耐药基因奠定基础.     

低水平乙型肝炎病毒表面抗原S区序列分析

目的对低水平乙型肝炎病毒表面抗原进行病毒S区基因分析。方法利用套式PCR法（Nested PCR）特异性扩增乙型肝炎病毒DNA的S区,对PCR扩增产物直接进行DNA序列分析,测序结果经Genotyping软件分型并与美国GeneBank中基因序列进行BLAST比对。结果 37例低水平乙型肝炎病毒表面抗原标本中,成功扩增并测序32份,其余5份扩增产物因不满足测序要求无法进行S区序列分析,32份成功测序标本经Genotyping分型,B型20例,C型12例,分别与GeneBank中收录的AF100309、AB014381同源性最高;血清学分型18例为adr,10例为adw,3例为ayw,1例为ayr,并发现21例S区核酸序列发生点突变,其中15例突变不会引起编码抗原决定簇氨基酸突变,为无义突变,而其他6例点突变均可引起抗原决定族编码的氨基酸的改变,从而导致表面抗原结构改变。结论对低水平乙型肝炎病毒表面抗原患者S区序列研究将为低水平乙型肝炎发病机制、流行病学以及个体化治疗奠定理论基础。     

Simple technique for internal control of real-time amplification assays

BACKGROUND: In real-time PCR assays, the most accurate way to identify false-negative results, e.g., those caused by PCR inhibitors, is to add to samples an internal control that will be coamplified with the target (e.g., pathogen) DNA. Current internal control procedures, however, which usually involve the introduction of a DNA fragment, are complex, time-consuming, and expensive. METHODS: Single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time PCR assays. Mismatches were included in the probe-binding region of the internal control oligonucleotide (ICO) to prevent probe-control hybridization during the fluorescence acquisition step of the PCR. Amplified ICOs were detected by melting point analysis. ICOs could be added directly to the sample material before DNA extraction. RESULTS: To demonstrate the feasibility of the new approach, we designed ICOs for the LightCycler hybridization probe assays for Mycobacterium tuberculosis complex, hepatitis B virus, herpes simplex virus, and varicella zoster virus. In each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis. CONCLUSIONS: A single-stranded oligonucleotide that mimics the target region of the pathogen but is clearly distinguishable from the target during melting point analysis can serve as a simple, cost-effective internal control for real-time amplification assays. Such control oligonucleotides are easy to design and inexpensive. A costly second probe system is not necessary. Moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications.     

A one-step dipstick assay for the on-site detection of nucleic acid

Objectives

We have developed a one-step nucleic acid dipstick assay (NADA) for visually detecting polymerase chain reaction (PCR) products within 3 min. “One-step” means that there were no additional procedures between amplification and detection.

Methods

This method was achieved through the use of asymmetric PCR and specially designed probes with appropriate melting temperature values. We initially combined one-step NADA with asymmetric capillary convective PCR (ACCPCR), an easy and rapid nucleic acid amplification technique, to construct an on-site nucleic acid diagnostic platform.

Results

We developed a diagnostic assay for the hepatitis B virus based on the ACCPCR-NADA platform to verify its feasibility. It exhibited an analytical sensitivity of three copies per test and a broad detection spectrum including genotype A–I. It also showed 97.9% sensitivity and 100% specificity based on the results observed using 67 serum samples with the Roche COBAS AmpliPrep/COBAS TaqMan (COBAS) system as the standard for comparison.

Conclusion

The results provide evidence for the feasibility of using an ACCPCR-NADA platform in practical applications, especially in on-site test.     

初诊慢肝患者血清中乙肝病毒DNA载量与血清标志物的关系

目的探讨初诊慢性乙型肝炎血清标志物与HBV DNA的关系。对象与方法采用酶联免疫方法和荧光定量聚合酶链反应分别对512例不同乙肝病人及202例HBeAg阴性、257例HBeAg阳性慢肝病人进行血清标志物、HBV DNA定量检测并分析结果。结果慢肝组有315例(68.9%)病毒颗粒浓度高于107,肝硬化组7例(13.2%)病毒颗粒浓度高于107;HBeAg阳性慢肝组245例(95.3%)患者病毒颗粒含量高浓度,HBeAg阴性慢肝组68例(33.7%)患者病毒颗粒含量在105-7copy/mL区段。结果慢肝组与肝硬化组相比,高浓度病毒以慢肝组中最明显,病情发展至肝硬化期病毒含量多数不高;慢肝病人中不同乙肝血清标志物模式其病毒含量分布特点不同,与HBeAg阳性慢肝组相比HBeAg阴性组体内病毒复制水平低(p<0.01),传染性较前者弱。最常见前C区突变,导致HBeAg阴性CHB病情加重和病毒复制增加。     

DNA微列阵技术检测戊型肝炎病毒方法的建立及初步应用

目的建立DNA微列阵技术检测戊型肝炎病毒方法,进而探讨该方法用于检测临床标本的可行性。方法通过生物医学数据库戊型肝炎病毒基因进行检索和筛选,应用分子生物学软件,进行序列分析、引物及探针设计,并进行验证,建立了戊型肝炎病毒DNA微列阵技术检测方法。结果试验所设计的探针仅与HEV的PCR产物杂交呈阳性,与乙肝、丙肝等对照病毒的PCR产物杂交呈阴性。敏感性试验显示,该方法比同巢式PCR方法检测戊型肝炎病毒敏感度要高,用该方法检测了长春地区50份疑似HEV临床病料,38份阳性;而用巢式PCR法扩增HEV ORF1基因确诊为阳性的只有35份。结论利用DNA微列阵技术检测戊型肝炎病毒方法,特异性和敏感性强,可作为HEV临床标本检测方法。     

Recent developments in the diagnosis and monitoring of HBV infection and role of the genetic variability of the S gene

Recent developments in the laboratory diagnosis of hepatitis B virus infection include the optimization of key serologic markers, including hepatitis B virus surface antigen and antihepatitis B virus core antibody, as well as the development of automated nucleic acid amplification assays. There is still a lack of standardization for nucleic acid amplification assays that are used for the monitoring of antiviral therapy and follow-up of chronic infection and the clinical significance of hepatitis B virus DNA levels need to be clarified. Although highly sensitive automated nucleic acid amplification assays for blood donor screening are available, their implementation is still subject to discussion and certain countries rejected hepatitis B virus DNA testing for blood donation due to poor cost effectiveness. Genetic variability of hepatitis B virus constitutes a major challenge for diagnosis of hepatitis B virus infection, particularly with regard to hepatitis B virus surface antigen detection, antihepatitis B virus surface antigen quantification and nucleic acid amplification assays. The performances of hepatitis B virus surface antigen enzyme immunoassays in regard to genotype and surface antigen variability need to be further improved. Polyclonal antibody-based hepatitis B virus surface antigen enzyme immunoassays, although they cannot guarantee 100% sensitivity, demonstrate superior S gene mutant recognition to assays using monoclonal capture and tracer antibodies. Isolated antihepatitis B virus core reactivity is an unusual but frequent result, which requires a test algorithm for resolution and hepatitis B virus DNA detection with sensitive nucleic acid amplification assays in order to exclude occult hepatitis B virus infection.     

Development of a novel protocol for RT-multiplex PCR to detect diarrheal viruses among infants and children with acute gastroenteritis in Eastern Russia

Viral gastroenteritis is one of the most common illnesses in humans worldwide and it has a great impact on people. Recently, we reported three RT-multiplex PCR assays termed A, B and C that could detect three groups of diarrheal viruses; group A, B and C rotaviruses and adenovirus [Phan et al., J Med Virol 2004; 74:173-9]; norovirus GI and GII, sapovirus and astrovirus [Yan et al., J Virol Meth 2003; 114:37-44]; enteroviruses, hepatitis A and E viruses and influenza A virus [Phan et al., Arch Virol 2005; 1175-85], respectively. In the present study, we developed a novel protocol with a small volume of reaction mixture for RT and PCR products (only 8 microl and 11 microl, respectively) that can amplify genomes of target viruses simultaneously. A total of 100 fecal specimens from infants and children with acute gastroenteritis in Birobiclzhan city, Eastern Russia, were collected during November 2003 and March 2004 and tested for the presence of those viruses by the novel RT-multiplex PCR protocol. Group A rotavirus was the most prevalent (67%), followed by norovirus GII (4%), group C rotavirus (1%), and sapovirus (1%). Interestingly, one fecal specimen turned out to be positive for hepatitis A virus. The sensitivity and specificity of RT-multiplex PCR assays with a novel protocol demonstrated a strong validation against the previously published RT-multiplex PCR. The findings clearly indicated that this novel protocol is simple and cost-effective to investigate the molecular epidemiology of acute gastroenteritis caused by diarrheal viruses. This report is the first, to our knowledge, detecting hepatitis A virus in feces from diarrheal infants and children in Eastern Russia.     

Genomic heterogeneity of hepatitis B virus in a 54-year-old woman who contracted the infection through materno-fetal transmission

From the plasma of a 54-year-old woman, who acquired the persistent carrier state of hepatitis B virus through materno-fetal transmission, 49 clones of viral genomes were propagated. They did not reveal any differences in the size and number of cleavage products with any of 11 restriction endonucleases. Randomly selected 5 clones were classified into 3 groups by the variation at 4 positions in the nucleotide sequence of the envelope and core genes. The complete nucleotide sequences were determined for 3 of them, each representing a group, and they all had a genomic length of 3215 nucleotides. Variation was found in from 5 to 11 nucleotides. Assuming the infection with the common ancestor virus at birth, hepatitis B virus genomes in her plasma were estimated to have evolved at a rate from 1.4 to 3.2 x 10(-5) nucleotide substitutions per site per year. This value is 10(4)-fold greater than DNA genomes, 10(2)-fold less than human immunodeficiency virus but in the same order as most RNA viruses including certain retroviruses.     

实时荧光聚合酶链反应检测HBV基因型方法的建立和临床研究

目的 建立实时荧光聚合酶链反应（PCR）检测乙型肝炎病毒（HBV）基因型方法并对检测的慢性乙型肝炎（CHB）患者基因型进行临床分析。方法 根据GenBank中已发表的明确分型的143株HBV全序列，设计特异的组合引物探针，建立实时荧光PCR方法，检测128例慢性乙型肝炎患者基因型；同时检测HBVDNA、HBV-M。结果 （1）128例标本中B型检出率为20．3％（26／128），C型占71．9％（92／128），D型占7．8％（10／128）；18株HBV克隆标本S基因测序结果与本分型法完全一致。（2）男性与女性的HBV基因型构成比无明显差异（P=0．561）。B型与C型比较，C基因型HBVDNA含量（P〈0．05）和HBeAg阳性率（P〈0．01）明显高于B基因型。基因型B具有更高的HBeAb水平（P〈0．05）。结论 （1）实时荧光聚合酶链反应HBV基因分型法能够简便、灵敏、快速、准确地鉴定HBV基因型。（2）HBV基因型主要以C型为主，其次为B型、D型。在CHB患者中，性别不是基因型构成差别的因素，C基因型易引起较重的肝脏病变，B型易发生免疫逃逸，致病情较轻。