多聚酶链反应——方法和仪器

<正>多聚酶链反应(PCR)类似体内DNA复制过程,在待扩增的“靶”或“模板”DNA双链分子的的两端各接一个引物,经一次循环后,可得到两个相同的双链靶DNA分子。DNA分子数随循环次数呈几何级数增长,30次循环后,靶DNA被放大2~(30)倍,其拷贝数可达10~9以上,在100ul PCB反应混合物中约产生2～3μ DNA。扩增的靶DNA分子长达10～18kb,但4kb抗更短的靶DNA最适宜PCR扩增,PCR能测出10~6基因组中的一个DNA拷贝。     

荧光定量PCR检测人巨细胞病毒的方法学建立

目的建立人巨细胞病毒（HCMV）的TaqMan MGB探针荧光定量PCR（FQ—PCR）检测方法。方法选取HCMV MIE exon4为PCR扩增靶序列,经TA克隆构建重组质粒作为定量标准品,经FQ—PCR反应条件的优化及方法学评价,再将其应用于临床检测。结果FQ—PCR最适循环参数为：95℃ 5 min;95℃ 20 s,60℃ 60 s（40 cycles）,20μl最适反应体系为：2．0mmol／L Mg^2＋、0．5μmol／L引物、1．5μmol／L探针、200μmol／L dNTP、2110×buffer、1．0 U Taq酶、2．0μl DNA模板。检测批内CV（变异系数）值为1．32％,批间CV值为1．96％;特异性较好;线性范围为10^2-10^8copies／μl。结论成功地建立了检测HCMV的FQ—PCR法,完全适用于临床检测。     

一种新的高效快速核酸恒温扩增方法--LAMP法

介绍一种新式恒温核酸扩增方法LAMP(Loop—Mediated Isothermal Amplification)法,其原理是采用四条特异引物及一种具有链置换活性的DNA聚合酶,在65℃左右对核酸进行扩增。短时间扩增效率可达到10^9-10^10个拷贝。LAMP法具有高特异性、高效性、快速、廉价、易检测等特点。预计在临床诊断、食品卫生检疫及微阵列基因芯片的开发上将有广阔的应用前景。     

标准品制备方法对FQ-PCR定量基因表达水平的影响

实时荧光定量PCR (FQ-PCR)标准曲线法准确定量基因表达的关键在于标准品与待检样本的扩增效率是否一致. 为检测DNA标准品与样本cDNA扩增效率的一致性,探讨定量用标准品的最佳制备方法,本研究以脂肪酸结合蛋白5(Fabp5)、过氧化物酶体增殖活化受体α (Ppar-α)及β肌动蛋白（β-Actin）的3个基因为对象,分别采用质粒纯化法、PCR产物直接纯化法、PCR产物凝胶回收法制备DNA标准品,10倍梯度稀释后用FQ PCR制作标准曲线. 并以10倍梯度稀释的样本cDNA标准曲线的参数为对照,进行比较分析. 结果表明,不同方法制备的DNA标准品的扩增效率差异较大,并且与cDNA的扩增效率不一致,不能对cDNA样本进行准确定量. 另外,虽然目的基因在cDNA样本中的拷贝未知,不能对基因表达水平进行绝对定量,但因不同cDNA样本的同一基因的扩增效率一致, 可对基因的表达进行准确的相对定量.     

荧光定量RT-PCR检测血管内皮细胞PAI-1 mRNA方法的建立

为了建立用荧光定量PCR检测人微血管内皮细胞纤溶酶原激活剂抑制物-1（PAI-1）mRNA表达的方法。提取人微血管内皮细总RNA,经RT-PCR获得靶基因（PAI-1）及管家基因（β-actin）的PCR产物。纯化后,作为标准品梯度稀释,采用SYBR Green I定量PCR检测,建立标准曲线。方法学考核参数为特异性、线性范围、灵敏性和重复性。分析全反式维甲酸对内皮细胞表达PAI-1 mRNA的干预效果。这种定量方法特异性好,检测的灵敏度达10^3拷贝,线性范围为10^4-10^10拷贝,循环阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系（r^2〉0.990）,批内变异≤4.93%,批间变异≤9.12%。全反式维甲酸能上调内皮细胞PAI-1 mRNA的表达,且呈剂量依赖性。因此,荧光定量RT-PCR检测血管内皮细胞PAI-1基因表达的方法有助于溶栓药物药理学的研究和新药筛选。     

SNP与基因检测及表达中的RCA技术

滚环扩增(rollingcircleamplification,RCA)技术是一种新的分子生物学检测方法。该方法不仅可以在体外等温条件下对核酸进行高度特异性的检测,而且还可通过线性或指数扩增来进行信号级联放大,其灵敏度能达到1个拷贝的核酸分子,因此,可用于痕量分子的检测。目前,滚环扩增技术广泛应用于全基因组DNA检测、核酸测序、单核苷酸多态性、DNA芯片及蛋白质芯片分析等领域。     

猴免疫缺陷病毒（SIV）实时荧光定量PCR检测方法的建立

目的建立快速、敏感、特异的猴免疫缺陷病毒（SIV）TaqMan探针实时荧光定量PCR检测方法,对SIV病毒核酸进行定量检测。方法RT—PCR扩增SIVmac251保守gag基因序列796bp片段,进行TA克隆,构建标准品质粒pMD—SIVgag。通过对SIV定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量PCR方法的灵敏度、特异性和重复性。结果所建立的SIVQPCR检测方法,质粒DNA模板在10’～10。拷贝之间表现较好线性和相关性,标准曲线所得斜率为-3．26,相关系数为0．999。检测灵敏度达到200拷贝,方法重复性测试,检测25份临床样品CV％均小于1％。结论建立的SIVQPCR检测方法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒（SIV）核酸拷贝量。     

RT-SHIV rt基因单拷贝PCR扩增方法的建立及初步应用

目的建立RT-SHIV病毒全长rt基因单拷贝PCR扩增方法,用于HIV-1 rt基因体内遗传与变异研究。方法 Oligo软件设计RT-SHIV rt基因特异性扩增引物,梯度稀释方法进行特异性和灵敏度筛选,进而优化退火温度和PCR反应最佳循环数等条件,建立rt基因PCR扩增方法;在此基础上将模板进行有限稀释,摸索rt基因单拷贝PCR扩增条件;使用该方法扩增感染猴体内RT-SHIV病毒rt基因,BioEdit软件进行基因序列分析。结果筛选得到一组巢式PCR引物,成功建立了RT-SHIV rt基因PCR扩增方法;当模板浓度为100 copies/μL时,扩增产物为单拷贝序列;测序结果显示RT-SHIV感染猴d266和d294血浆样本分别存在1处和6处氨基酸突变。结论本研究建立的全长rt基因单拷贝PCR扩增方法特异性好、灵敏度高、重复性强,可以应用于各类RT-SHIV病毒的全长rt基因分析。     

逆转录环介导等温核酸扩增技术(RT-LAMP)在H5N1禽流感病毒基因检测中的应用

建立一种便捷、灵敏的检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)用于H5N1亚型禽流感病毒基因检测.该技术使用特异对应于靶序列中8个基因区段的6条特异引物,在等温条件下进行核酸扩增反应.对51份实验感染动物及病毒培养标本的H5N1亚型禽流感病毒的HA、NA基因区进行了RT-LAMP检测,并以SYBR Green Ⅰ为反应指示剂进行了逆转录环介导等温核酸扩增技术,对该反应进行实时监控,经对扩增产物做内切酶验证和测序分析,证明RT-LAMP技术的特异性;同时,用10倍系列稀释的RNA样品对该检测方法的灵敏度进行了测试.结果显示:利用RT-LAMP技术成功检测到H5N1禽流感病毒的HA、NA基因区,且RT-LAMP与Real-time PCR结果呈现很好的一致性.此方法的灵敏度可达到能检测10个拷贝RNA分子水平.因此,RT-LAMP技术应用于H5N1亚型禽流感病毒的快速检测是一种可行的方法.     

聚合酶链反应（PCR）对未知序列DNA的扩增技术

聚合酶链反应(Polymerase Chain Reaction, PCR)是由引物介导,在体外将特异性DNA序列利用酶促作用进行扩增的方法。双链DNA热变性,然后在低温下与引物退火,再在中等温度下进行延伸,三步为一循环。一般经30～35次循环,很容易将目的基因或DNA片段特异地扩增至少10~6～10~7倍。它已是分子生物学研究中常用的、不可缺少的一项基本技术。但常规PCR需在待扩增的已知序列两端分别设计两个寡核苷酸引物,也就     

HBV X 区基因部分序列的亚克隆与PCR 检测

目的：探讨HBVX区基因用于乙型病毒性肝炎早期诊断价值和意义。方法：设计特异性引物,将pBR322-HBV质粒X区部分序列PCR产物AT亚克隆至pBS—T载体,提取和纯化质粒DNA,再对HBVX区序列进行PCR扩增。结果：获得了预期希望的质粒。PCR的最佳退火温度为51℃,灵敏度达到101拷贝／2μl,线性范围101—1010拷贝／2μl。讨论：pBR322-HBV质粒中的靶基因成功地被亚克隆至pBS—T载体。pBR322-HBV中X区目的序列扩增产物为57bp,该小片段勿需纯化就可直接AT亚克隆至新载体,有利于后续的常规PCR检测和TaqMan MGB荧光定量PCR检测。     

Potential influence of the first PCR cycles in real-time comparative gene quantifications

There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1-3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.     

The PCR plateau phase - towards an understanding of its limitations

The DNA polymerases from Thermus aquaticus and Thermus flavus were recently found to bind to short double-stranded DNA fragments without sequence specificity [Kainz et al. (2000) Biotechniques 28, 278-82]. In the present study, it is shown that the accumulation of amplification products during later PCR cycles also exerts an inhibitory effect on several enzymes tested. To simulate later cycle conditions, a 1.7 kb sequence from phage lambda DNA was amplified in the presence of various amounts of a 1 kb double-stranded DNA fragment. A 30-fold molar excess of fragments to polymerase molecules was found to be required for a complete inhibition of Taq, Tfl and Pwo DNA polymerase. This stoichiometric relation remained constant when PCR amplifications were performed using polymerase concentrations of 0.5, 1 or 1.5 U/50 microl reaction volume. The amount of 1 kb DNA fragments required for a complete inhibition was similar to the product yield of the controls (no fragment added), that were run to plateau phase levels. Additionally, PCR mixtures, that were subjected to different numbers of cycles, were compared in their ability to extend 3'-recessed ends by using a hairpin extension assay. The presence of endogenous amplicon DNA accumulated in later PCR cycles was found to inhibit completely the activity of DNA polymerase. PCR mixtures still in quasi-linear phase partially extended the hairpins. In both cases, a further addition of polymerase significantly improved their function. These results indicate that the main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products.     

基孔肯雅病毒和辛德毕斯病毒可视化基因芯片与荧光基因芯片的建立

根据GenBank中收录的基孔肯雅病毒和辛德毕斯病毒E蛋白基因序列,设计及筛选针对2种病毒的寡核苷酸探针及引物,制备基孔肯雅病毒与辛德毕斯病毒可视化基因芯片与荧光基因芯片,对芯片的灵敏性、特异性进行了验证,并将可视化基因芯片、荧光基因芯片进行灵敏性比较.结果显示,制备的两种基因芯片都能检测到基孔肯雅病毒和辛德毕斯病毒特异性杂交信号.可视化基因芯片、荧光基因芯片检测两种病毒质粒的灵敏度达到9.1×103 copies/mL, 6.8×101 copies/mL和9.1×104 copies/mL, 6.8×103 copies/mL,与普通PCR比较差异显著. 荧光基因芯片灵敏度是PCR方法的10倍,可视化基因芯片是荧光基因芯片灵敏度的100倍. 模拟病毒检测过程特异性检验证明,可视化基因芯片都具有良好的特异性.本试验建立了基孔肯雅病毒与辛德毕斯病毒两种特异的可视化和荧光基因芯片检测方法,两种方法灵敏度高、特异性强,适用于基孔肯雅病毒与辛德毕斯病毒的流行病学调查和种特异性鉴定.     

Variation in the molecular mass of the Ah receptor among vertebrate species and strains of rats

The Ah receptor in eight vertebrate species was characterized by labeling the cytosolic fraction of tissue with the photoaffinity ligand, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, and analysis of the products by denaturing gel electrophoresis. The apparent molecular mass of the dominant labeled peptide showed appreciable species variation: mouse-95 kDa; chicken (embryo)-101 kDa; guinea pig-103 kDa; rabbit-104 kDa; rat-106 kDa; human-106 kDa; monkey-113 kDa, and hamster-124 kDa. Seven inbred strains of rats, had a Ah receptor ligand binding peptide of 106 kDa; however outbred Long-Evans rats were shown to be polymorphic expressing a 101 kDa and/or 106 kDa allelic forms. The notable frequency of structural variation in the Ah receptor is in contrast to the analogous highly conserved steroid hormone receptors.     

Solid-phase time-resolved fluorescence detection of human immunodeficiency virus polymerase chain reaction amplification products.

A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.     

3种菊花病毒/类病毒实时荧光定量RT-PCR检测体系的构建与准确性评价

菊花容易受到病毒感染而造成品质下降,目前国内对菊花病毒的检测主要根据外观表现或者定性PCR检测,无法准确判定病毒载量。为构建一种可同时用于检测菊花B病毒(Chrysanthemum virus B,CVB)、番茄不孕病毒(Tomato aspermy virus,TAV)和菊花褪绿斑驳类病毒(Chrysanthemum chloritic mottle viroid,CChMVd)的实时荧光定量RT-PCR检测方法,本研究分别以保守区域作为靶标设计相应的引物探针,通过优化扩增体系中CVB、TAV、CChMVd 3种病毒/类病毒探针浓度、引物浓度、Mg²⁺浓度、dNTPs浓度,摸索扩增程序中反转录时间、退火温度和扩增循环数,构建了一种可同时用于CVB、TAV、CChMVd的3重实时荧光定量RT-PCR检测体系,优化后的扩扩增体系中CVB、TAV和CChMVd的探针浓度分别为100 nmol/L、120 nmol/L和80 nmol/L,引物浓度分别为200 nmol/L、240 nmol/L和160 nmol/L,Mg²⁺浓度为3.0 mmol/L;dNTPs浓度200μmol/L;最适反转录时间为25 min,退火温度为60℃,循环数为40。敏感性实验结果表明,该反应体系对3种病毒/类病毒的敏感性为1.0×¹⁰3拷贝/mL,敏感性好;定量线性范围为1.0×¹⁰3拷贝/mL~1.0×¹⁰10拷贝/mL,线性范围宽;特异性好,对菊花矮化类病毒、烟草花叶病毒和黄瓜花叶病毒核酸检测结果为阴性;对1.0×¹⁰4拷贝/mL的低浓度参考品平行检测10次,定量结果lg值偏差(CV%)为4.81%,重复性好。在南京农业大学"中国菊花种质资源保存中心"基地随机选择菊花20株进行本研究试剂检测,检出6例CVB病毒株和4例TAV病毒株,其病毒载量为2.5×¹⁰4拷贝/mL~5.5×¹⁰7拷贝/mL,随机选择1株CVB病毒株定量PCR,产物进行TA克隆后经测序与NCBI Blast比对,其与MH678704.1的同源性为100%。因此,本研究建立了一种能同时检测CVB、TAV、CChMVd 3种菊花常见病毒/类病毒的灵敏、快速、可定量的检测方法。     

PCR法对鸡卵黄中Coxiella burnetii菌的测定

[目的]通过用定量PCR加巢式PCR方法,提高了对Coxiella burnetii (C.b)CoMl基因的检出率;通过对鸡卵中病原微生物Coxiella burnetii的基因检测,明确鸡卵的食品安全性;并对明确Coxiella burnetii的流行病学有重要意义.[方法]提取鸡卵DNA,用定量PCR加巢式PCR方法检测上述基因,并对PCR产物进行测序分析,通过间接免疫荧光法观察鸡血白细胞中的微生物.[结果]用定量PCR加巢式PCR方法可检出4个以上的Coxiella burnetii Coml基因,用此方法可测出鸡卵中Coxiella burnetii Coml基因达104-106个,阳性率为5％-22％;对阳性鸡卵Coml基因PCR产物的测序结果显示有变异菌株的存在;免疫荧光法可见鸡卵中含有该微生物.[结论]由此认为鸡卵中存在病原微生物Coxiella burnetii,可能是Q热传染源.     

PCR产物中无错误碱基拷贝最低比率的计算

分析了PCR过程中带有错误碱基拷贝的量变过程,得出不同循环(n)后不同类型拷贝数的计算通式并以逐次代入方式归纳出PCR产物中无错误碱基拷贝最低比率(R)和有效循环数(N),拷贝酶促合成链长(H)及错配率(f)的关系式R_n=(1-Hf/2)^N-1(1-Hf),对PCR技术制备表达用DNA片段有指导意义.