Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.     

A methodological basis for description and analysis of systems with complex switch-like interactions

 A wide range of complex systems appear to have switch-like interactions, i.e. below (or above) a certain threshold x has no or little influence on y, while above (or below) this threshold the effect of x on y saturates rapidly to a constant level. Switching functions are frequently described by sigmoid functions or combinations of these. Within the context of ordinary differential equations we present a very general methodological basis for designing and analysing models involving complicated switching functions together with any other non-linearities. A procedure to determine position and stability properties of all stationary points lying close to a threshold for one or several variables, so-called singular stationary points, is developed. Such points may represent homeostatic states in models, and are therefore of considerable interest. The analysis provides a profound insight into the generic effects of steep sigmoid interactions on the dynamics around homeostatic points. It leads to qualitative as well as quantitative predictions without using advanced mathematical methods. Thus, it may have an important heuristic function in connection with numerical simulations aimed at unfolding the predictive potential of realistic models. Received 25 January 1996; received in revised form 29 June 1997     

乙状结肠癌经腹膜内造瘘与经腹膜外造瘘的临床疗效及并发症的预防

目的:探讨乙状结肠经腹膜内造瘘与经腹膜外造瘘的对比分析及造口并发症的预防。方法:选取我院收治的行肛管直肠下段恶性肿瘤切除术的患者127例,根据造瘘方式不同将患者分为两组,实验组72例予以经腹膜外造瘘,对照组55例予经腹膜内造口术,观察并比较两组患者的术中情况,术后并发症,以及术后排便功能,来评价两种造瘘方法的效果。结果:与对照组比较,实验组术中、术后并发症均降低,术后排便功能改善显著,差异均有统计学意义(P0.05)。结论:经腹膜外乙状结肠造口较经腹膜内乙状结肠造口的术中情况明显改善,且并发症减少,能够较好的控便和排便,值得临床推广使用。     

Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment

A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.     

The effect of redox potential on the kinetics of fluorescence induction in pea chloroplasts. II. Sigmoidicity

The shape of the fluorescence induction curve in chloroplasts inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea has been determined at different redox potentials. At ?10 mV a monophasic and sigmoidal curve is seen which is transformed into an exponential curve when the potential is poised at ?150 mV. At this potential, the quencher with high midpoint, Q_H, is reduced but that with low midpoint, Q_L, is oxidized. Thus, a sigmoidal induction is observed during photoreduction of Q_L and Q_H but photoreduction of Q_L proceeds with exponential kinetics. A correlation between the relative proportions of Q_L and Q_H observed in redox titration and the sigmoidicity of induction is also seen upon depletion of Mg²⁺ and after alkalinization to pH 9.5. Several models are discussed to explain the relationship between Photosystem II interactions and Q heterogeneity.     

乙状结肠扭转的CT表现

目的:乙状结肠扭转既往主要依靠腹部平片进行诊断,但腹部平片具有局限性,容易误诊、漏诊,CT可以弥补腹平片不足.探讨乙状结肠扭转的CT征象,以提高其术前诊断水平.方法:回顾性分析手术中证实为乙状结肠扭转的23例患者,分析其腹部CT扫描图和横断面扫描特征,最后总结乙状结肠扭转的CT影像学特征.结果:在CT定位像上,最敏感的表现是扩张的乙状结肠倒U征(21/23,91％),不成比例乙状结肠扩张(19/23,83％),其次为咖啡豆征(17/23,74％),Y征(12/23,52％).在横断面扫描上,最敏感的是不成比例的乙状结肠扩张(21/23,91％)和圆腹征(20/23,87％),其次为近端结肠扩张(15/23,65％),漩涡征(15/23,65％),鸟嘴征(13/23,57％).在CT定位像和横断面扫描上,典型的乙状结肠扭转影像表现分别占57％(13/23)和83％(19/23).在定位像上,咖啡豆征与Y征在显著相关(p＜0.05).横断面扫描上肠管损害的CT征象与临床肠管损害显著相关(P＜0.05).结论:在定位像上,乙状结肠扭转的主要影像学表现为倒U征,特征性表现为Y征;在横断面扫描上,主要影像学表现为不成比例的乙状结肠扩张,特征性表现为漩涡征和鸟嘴征.多排螺旋CT是诊断乙状结肠扭转的可靠检查方法,具有重要的临床应用价值.     

A Theoretical Basis for Gompertz'S Curve

GOMPERTZ'S model (1825) has remained a purely empirical one, despite its frequent usage. A theoretical justification is given which permits its employment in cases of accretionary growth.