青檀SRAP-PCR体系优化设计方案

以青檀(Pteroceltis tatarinowii Maxim.)叶片为材料,采用正交设计和均匀设计两种方法对SRAP-PCR反应体系进行优化,并对这两种设计方案优化出的最佳反应体系进行比较,结果表明:两种设计均可用于青檀SRAP-PCR体系的优化,但与正交设计相比,均匀设计在多因素多水平条件下,得到的条带更清晰、稳定性更好。通过实验比较筛选出的青檀SRAP-PCR最佳反应体系为:2.5μL 10×PCR buffer,20 ng模板DNA,Mg2+2.5 mmol/L,dNTP150μmol/L,引物0.2μmol/L,Taq DNA聚合酶1.0 U,总体积25μL。     

应用降落PCR和正交设计优化AtSUC9 PCR反应体系

目的:建立最适的AtSUC9 PCR反应体系。方法:应用降落PCR和正交设计,从Mg2 、dNTPs、引物、甘油4种因素3个水平,对拟南芥AtSUC99 PCR反应体系进行优化。结果:确立了适合拟南芥AtSUC99 PCR反应体系:在20μl反应体系中,含模板1μg,引物(20μmol/L)0.5μl,dNTPs(10mmol/L)0.3μl,10×PCRbuffer2μl,Mg2 (25mmol/L)1.2μl,TaqDNAPolymerase1U,甘油10μl。结论:降落PCR和正交设计是一种有效的植物基因PCR体系优化方法。     

星星草RAPD-PCR反应体系建立与优化

目的:寻找一个可用于星星草(Puccinellia tenuiflora(Griseb.)Scribn.et Merr)RAPD-PCR的最适宜的反应体系。方法:利用正交设计法对影响PCR扩增效果的一些因素诸如TaqDNA聚合酶的用量、Mg2 浓度、dNTP以及引物浓度等指标进行筛选和优化。然后,通过引物对该优化结果进行验证。结果:正交设计结果表明,最适宜的PCR反应条件:20μl PCR反应体系中包括10×buffer2μl、模板DNA20ng、dNTP1.6mmol/L、TaqDNA聚合酶0.35U、引物1.0mmol/L、Mg2 1.8mmol/L。验证结果表明,该体系扩增出的条带多且清晰。结论:该研究得出的体系是适合RAPD-PCR的最适宜的反应体系,为今后星星草遗传多样性的研究奠定了基础。     

食源性致病菌多重PCR快速检测方法建立与应用

利用PCR技术,建立多组多重食源性致病菌PCR快速检测方法。设计受试菌特异性引物,反应体系中加入多对引物和多种DNA模板,采用正交试验优化PCR反应条件,进行特异性引物的PCR扩增。建立了多组多重食源性致病菌PCR快速检测方法,方法中所检测受试菌株和模拟样品均出现特异性扩增条带,结果与实际相符。所建立多组多重PCR快速检测体系符合设计要求,可以应用于食源性突发公共卫生事件的应急检测和日常样品检测工作。     

D3S1358、D21S11和FGA复合扩增系统的研究

目的：研究D3S1358、D21S11和FGA3个STR基因座复合扩增的最佳体系。方法：设计正交实验优化复合扩增最佳反应条件,然后采用聚丙烯酰胺凝胶电泳分离显带技术检测扩增片段。结果：获得复合扩增条件各反应因素较为满意的参数。     

CYP2C19基因多态性PCR扩增体系的正交优化与验证

本实验通过建立CYP2C19*2、*3和*17基因多态性PCR反应体系和条件,筛选出4μL体系中各因素最佳水平。采用SNaPshot技术对CYP2C19基因3个SNP位点*2、*3和*17同时进行复合扩增检测,利用L9(34)正交实验设计,对影响PCR反应体系和条件的3个因素(PCR Mix, Taq DNA聚合酶,循环次数)在3个水平上进行优化,结果采用综合评分法和极差分析法进行分析。用3组已知样本对正交优化所得条件进行重复性和稳定性验证。结果表明CYP2C19*2、*3和*17基因PCR扩增体系的影响因素依次为:PCR Mix>循环数>Taq DNA聚合酶。最佳反应体系为PCR Mix 2.0μL、Taq DNA聚合酶0.2μL、循环次数32次。3组样本验证效果满意。优化的CYP2C19*2、*3和*17基因PCR反应体系稳定性高,重复性和经济性较好,为该基因多态性的大规模调查奠定了基础。     

利用MPprimer设计引物并优化扩增条件以提高多重PCR效率的实验研究

多重PCR技术广泛应用于多个研究领域,其中引物设计及扩增条件是提高多重PCR实验效率的关键因素.为探讨优化多重PCR实验的方法,以小鼠5个看家基因为研究对象,使用实验室新近开发的MPprimer程序设计多重PCR引物,并通过改变多种反应条件来优化多重PCR实验.结果表明,MPprimer程序能够设计出理想的多重PCR引物,并且通过对退火温度及延伸时间进行优化,可显著提高多重PCR实验效率,对于提高基因表达的规模化检测能力具有积极的促进作用.     

正交法整体优化差异显示反应体系

mRNA 差异显示PCR( mRNA differential display PCR,DDRT-PCR)是分离差异表达基因的有效方法,但该方法的准确性极易受到外部因素和内部因素的影响。本研究采用正交法优化DDRT-PCR反应条件,充分考虑到模板浓度、锚定引物浓度、随机引物浓度、dNTPs浓度、镁离子浓度以及Taq酶用量等因素在差异显示反应过程中的交互作用,一次PCR反应即可确定最佳反应组合。将筛选出的条件用于DDRT-PCR,得到差显结果假阳性率低,重复性和稳定性好,而且简化了反应条件的优选程序,这表明正交法是优化差异显示反应条件的理想方法。为了进一步简化整个差异显示反应系统的操作程序,降低假阳性率,研究采用了非变性聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)和银染显示差异带的方法,并用反向Northern法来验证回收条带,从而更加优化了差异显示反应体系。     

正交设计优化东亚砂藓DDRT-PCR反应体系

利用正交实验设计L25（5^6）对东亚砂藓（Racomitrium japonicum）DDRT—PCR反应体系的6因素（Mg^2＋、dNTP、锚定引物、随机引物、模板DNA、Taq酶）在5个水平上进行优化实验。结果筛选出各反应因素的最佳体系（20μL）为：Mg^2＋2．25mmol／L、dNTP0．4mmol／L、锚定引物1．0μmol／L、随机引物0．7μmol／L、模板DNA1．6μL、Taq酶2．5U。对东亚砂藓DDRT—PCR最佳反应体系进行梯度PCR引物退火温度筛选,得到的最佳退火温度为45．4℃。该优化体系的建立,为进一步进行东亚砂藓抗旱基因的筛选与克隆等一系列分子研究提供了重要参考依据。     

优化萝卜基因组DNA RAPD-PCR反应体系的正交设计法

以CTAB微量提取方法提取10个红萝卜雄性不育系A单株的基因组DNA,等浓度混合成基因池。以其为模板,用正交设计方法论定RAPD-PCR反应体系的各影响因子,用随机引物S42（5’GGACCCAACC3’）优化萝卜雄性不育系A基因组DNARAPD,PCR反应体系,16次试验即可获得最佳的RAPD-PCR反应体系。     

Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube

Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.     

正交优化法建立奶牛基因组DNA RAPD-PCR最佳反应体系

从 36头荷斯坦奶牛血样中提取基因组DNA ,以相同DNA浓度混成DNA池。以其为模板 ,通过正交设计试验 ,建立了RAPD PCR最佳反应体系。     

Pre-PCR processing

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.     

Optimal nonsingular control of fed-batch fermentation

Presented is a new simple method for multidimensional optimization of fed-batch fermentations based on the use of the orthogonal collocation technique. Considered is the problem of determination of optimal programs for fermentor temperature, substrate concentration in feed, feeding profile, and process duration. By reformulation of the state and control variables is obtained a nonsingular form of the optimization problem which has considerable advantage over the singular case since a complicated procedure for determination of switching times for feeding is avoided. The approximation of the state variables by Lagrange polynomials enables simple incorporation of split boundary conditions in the approximation, and the use of orthogonal collocations provides stability for integration of state and costate variables. The interpolation points are selected to obtain highest accuracy for approximation of the objective functional by the Radau-Lobatto formula. The control variables are determined by optimization of the Hamiltonian at the collocation points with the DFP method. Constraints are imposed on state and control variables.The method is applied for a homogeneous model of fermentation with volume, substrate, biomass, and product concentrations as the state variables. Computer study shows considerable simplicity of the method, its high accuracy for low order of approximation, and efficient convergence.     

A two-step quantitative pathogen detection system based on capillary electrophoresis

Rapid identification of bacterial pathogens is important for patient management and initiation of appropriate antibiotic therapy in the early stages of infection. Among the several techniques, capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis combined with small subunit rRNA gene-specific polymerase chain reaction (PCR) has come into the spotlight owing to its sensitivity, resolution, and reproducibility. Despite the advantages of the method, the design of PCR primers and optimization of multiplex PCR conditions remain to be studied so that as many pathogens as possible can be analyzed in a single run. Here we describe a novel two-step technique involving multiplex PCR pathogen detection by CE-SSCP analysis followed by singleplex PCR pathogen quantification by CE-SSCP. Specific PCR primers were designed for optimal separation of their products by CE-SSCP based on molecular weight. PCR conditions were then optimized for multiplex analysis of the targets. Subsequently, detected pathogens were quantified by PCR with specific primers. Eight clinically important strains were simultaneously identified under the optimized conditions. Each individual pathogen was then quantified at a level of sensitivity of tens of cells per milliliter. In conclusion, the two-step pathogen detection method based on CE-SSCP described here allows for sensitive detection of pathogens by multiplex PCR (first step) and quantification by specific PCR (second step). The results illustrate the potential of the method in clinical applications.     

高分辨率熔解曲线法检测CYP4A118590T＞C单核苷酸多态性

目的 建立CYP4A11 8590T＞C单核苷酸多态性（single nucleotide polymorphism,SNP）的高分辨率熔解曲线（high resolution melting,HRM）检测方法.方法先采用温度梯度PCR,确定适宜的退火温度;再利用正交试验,优化引物、DNA模板量和Mg2＋量,最终确定PCR反应体系和反应条件.通过对607例无血缘关系的受试者基因组DNA进行HRM分析,并随机选择50例产物测序.结果 引物最适退火温度为57.8 ℃;PCR最佳反应体系为20 μl,包括2×conc dNTP mix 10 μl,上下游引物（10 μmol/L）各0.5 μl,DNA溶液（30 ng/μl）1.0 μl,Mg2＋（2.5 mmol/L）1.5 μl和灭菌水6.5 μl.607例受试者中CYP4A11 8590TT、TC和CC基因型频率分别为54.7 %、37.6 %和7.7 %.结论该正交试验优化的HRM技术可用于检测CYP4A11 8590T＞C单核苷酸多态性,且其分析结果和测序结果一致.     

Problems with and a system to eliminate single-primer PCR product contamination in simple sequence repeat molecular marker-assisted selection in soybean

Polymerase chain reaction (PCR) provides a foundation for simple sequence repeat molecular marker-assisted selection (SSR MAS) in soybean. This PCR system and its various conditions have been optimized by many researchers. However, current research on the optimization of the PCR system focuses on double-primer PCR products. We compared single- and double-SSR primer PCR products from 50 soybean samples and found that the use of single-PCR primers in the reaction system can lead to amplified fragments of portions of the SSR primers in the PCR process, resulting in both false-positives and fragment impurity of double-primer PCR amplification, inconvenient for subsequent analysis. We used "single-primer PCR correction" to eliminate interference caused by single-primer nonspecific PCR amplification and improve PCR quality. Using this method, the precision and success rates of SSR MAS in soybean can be increased.     

A Fast-and-Robust Profiler for Improving Polymerase Chain Reaction Diagnostics

Polymerase chain reaction (PCR) is an in vitro technology in molecular genetics that progressively amplifies minimal copies of short DNA sequences in a fast and inexpensive manner. However, PCR performance is sensitive to suboptimal processing conditions. Compromised PCR conditions lead to artifacts and bias that downgrade the discriminatory power and reproducibility of the results. Promising attempts to resolve the PCR performance optimization issue have been guided by quality improvement tactics adopted in the past for industrial trials. Thus, orthogonal arrays (OAs) have been employed to program quick-and-easy structured experiments. Profiling of influences facilitates the quantification of effects that may counteract the detectability of amplified DNA fragments. Nevertheless, the attractive feature of reducing greatly the amount of work and expenditures by planning trials with saturated-unreplicated OA schemes is known to be relinquished in the subsequent analysis phase. This is because of an inherent incompatibility of ordinary multi-factorial comparison techniques to convert small yet dense datasets. Treating unreplicated-saturated data with either the analysis of variance (ANOVA) or regression models destroys the information extraction process. Both of those mentioned approaches are rendered blind to error since the examined effects absorb all available degrees of freedom. Therefore, in lack of approximating an experimental uncertainty, any outcome interpretation is rendered subjective. We propose a profiling method that permits the non-linear maximization of amplicon resolution by eliminating the necessity for direct error estimation. Our approach is distribution-free, calibration-free, simulation-free and sparsity-free with well-known power properties. It is also user-friendly by promoting rudimentary analytics. Testing our method on published amplicon count data, we found that the preponderant effect is the concentration of MgCl₂ (p<0.05) followed by the primer content (p<0.1) whilst the effects due to either the content of the deoxynucleotide (dNTP) or DNA remained dormant (p>0.1). Comparison of the proposed method with other stochastic approaches is also discussed. Our technique is expected to have extensive applications in genetics and biotechnology where there is a demand for cheap, expedient, and robust information.     

Optimization of PCR in application of hot start Taq DNA polymerase for detection of Erwinia amylovora with primers FER1-F and FER1-R

There are two approaches in detection of bacterium Erwinia amylovora by PCR. One is based on detection of plasmid pEA29 and the other is based on detection of a chromosomal DNA sequence, specific for E. amylovora, in a sample. Since pathogenic strains without pEA29 have been isolated from the environment, methods based on this plasmid have been compromised and PCR methods based on chromosomal DNA species specific sequences became only reliable methods. PCR method with chromosomal primers FER1-F and FER1-R is currently the most reliable method due to its high sensitivity and specificity. The goal of this research is to make a significant improvement of the method by optimization of PCR in application of hot start DNA Taq polymerase, instead of wax, to obtain a hot start reaction. This enzyme, which is currently widely applied, can provide simpler achievement of hot start, saving labor and time and decreasing possibility of cross contamination of samples. Experiments showed that simple replacement of a regular recombinant Taq DNA polymerase by a hot start Taq DNA polymerase leads to complete failure of the reaction. Many optimization experiments had to be carried out to obtain an operational and reliable PCR which simultaneously has high sensitivity and specificity. Content of the reaction mixture, as well as temperature and time parameters of PCR, were significantly changed to achieve proper optimization.