Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features.

Primary cultures of rat fetal hepatocytes were used to study the effect of insulin on glycogenesis during 24 h. Measurements of both glycogen content and 14 C-glucose incorporation into glycogen gave similar results. A clear stimulatory insulin effect was observed within one hour, reaching a maximum after three hours. However, from 4 to 12 h, the effect declined strongly and then disappeared. Thereafter, it reappeared but to a lesser extent. These striking variations were not caused by a lack of active insulin in the medium or by any amplification of time variations in hepatocyte basal glycogenic capacity. They were induced by the hormone itself as a function of time of insulin presence at the hepatocyte level. Therefore, the effect of insulin is time dependent.The transient cessation in the stimulatory effect of insulin on net glycogenesis limits the total amount of glycogen stored and permits a more progressive glycogen accumulation. It is not yet clear whether this cessation is a result of a decrease in the insulin-stimulated glycogen synthesis, or an increase of glycogen breakdown, or both; but we showed that it does not require prior maximal stimulation of glycogenesis. After four hours of incubation with varying doses of insulin, a dosedependent decrease in the insulin glycogenic effect of a second maximal dose of hormone was observed, whereas glucagon-induced glycogenolysis was unchanged. The characteristics of the time dependence of the insulin effect suggest that it may play a regulatory role in fetal hepatocytes. DIABETES 28:705-712, August 1979.H epatocytes isolated from adult rat liver, maintained in survival or, more recently, in primary culture, were used to study the direct regulatory effects of insulin on cell metabolism. The rapid effects of insulin, such as the stimulation of amino-acid uptake, are well documented. 1 -2 Slower processes, such as the induction of omithine transcarbamylase by insulin, have been described using cultured hepatocytes. 3 Concerning the specific role of insulin on carbohydrate metabolism, rapid activation of glycogen synthase and glycogenesis were observed in a few cases with isolated hepatocytes. 4 " 5 In cultured adult hepatocytes, a clear glycogenic effect of insulin was obtained, but it required an important lagtime. 6 However, we have shown that cultured fetal hepatocytes respond to insulin within four hours by giving a large increase in glycogen content. 7 The aim of the present study was to examine the glycogenic effect of insulin in this fetal system as a function of time. MATERIALS AND METHODSPrimary cultures of hepatocytes were obtained from 18-dayold Sprague-Dawley rat fetuses, a stage characterized in vivo by the first deposits of glycogen in rat hepatocyte. 8 Culture system. The culture procedure, which has already been described, 9 eliminates the hematopoietic population of the fetal liver. More than 90% of the cells are typical hepatocytes, the remaining 10% being fibroblast-like cells. The composition of the culture medium was as follows: NCTC 109, 90 p...

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