生产-流通关系协调发展：理论与对策研究

在流通产业对现代生产发展的意义日益加大的同时,我国传统流通产业发展滞后于生产发展的问题也日益暴露。模块化时代的到来,生产、流通环节的专业化分工要求有更多的协作,分工的协调效率受到更多的关注。目前我国生产-流通关系不协调,原因主要在于流通产业发展滞后于生产需要,借助于产业链整合实现流通产业的规模化发展,提升专业化流通渠道的效率功能,是中国产业系统健康发展的有效途径。      

Ultrafast Amplification of DNA on Plastic Microdevices for Forensic Short Tandem Repeat Analysis

The majority of microfluidic devices used as a platform for low‐cost, rapid DNA analysis are glass devices; however, microchip fabrication in glass is costly and laborious, enhancing the interest in polymeric substrates, such as poly (methyl methacrylate) (PMMA), as an inexpensive alternative. Here, we report amplification in PMMA polymerase chain reaction (PCR) microchips providing full short tandem repeat profiles (16 of 16 loci) in 30–40 min, with peak height ratios and stutter percentages that meet literature threshold requirements. In addition, partial profiles (15 of 16 loci) were generated using an ultrafast PCR method in 17.1 min, representing a ~10‐fold reduction in reaction time as compared to current amplification methods. Finally, a multichamber device was demonstrated to simultaneously amplify one positive, one negative, and five individual samples in 39 min. Although there were instances of loci dropout, this device represents a first step toward a microfluidic system capable of amplifying more than one sample simultaneously.     

High‐Throughput STR Analysis for DNA Database Using Direct PCR,

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high‐throughput and cost‐effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter‐loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time‐ and cost‐saving manner.     

A Molecular Key for the Identification of Blow Flies in Southeastern Nebraska*

Abstract: Immature blow flies (Calliphoridae) are typically the first colonizers of cadavers. Identification of the early instars using traditional, morphology‐based keys is difficult because of their small size, similarity, and simplicity in external morphology. Information derived from molecular genetic data would augment the accurate identification of immature flies. Nine species of blow flies commonly found in southeastern Nebraska were used to examine the utility of molecular‐based keys. Polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLP) were investigated with 10 common, inexpensive, restriction enzymes from an amplicon of approximately 1500 bp spanning the mitochondrial cytochrome oxidase I gene. A simple molecular taxonomic key, comprising RFLP from the restriction enzymes HinfI and DraI, enabled the differentiation of all species used. Further development of PCR–RFLP, including more extensive and intensive examination of blow flies, would benefit forensic laboratories in the accurate identification of evidence consisting of immature blow flies.     

Simple and Sensitive Method for Identification of Human DNA by Allele-Specific Polymerase Chain Reaction of FOXP2

Abstract:  The forkhead box P2 ( FOXP2 ) gene is specifically involved in speech and language development in humans. The sequence is well conserved among many vertebrate species but has accumulated amino acid changes in the human lineage. The aim of this study was to develop a simple method to discriminate between human and nonhuman vertebrate DNA in forensic specimens by amplification of a human-specific genomic region. In the present study, we designed an allele-specific polymerase chain reaction (PCR) using primers to amplify smaller than 70-bp regions of FOXP2 to identify DNA as being of human or nonhuman, including ape, origin. PCR amplification was also successfully performed using fluorescence-labeled primers, and this method allows a single PCR reaction with a genomic DNA sample as small as 0.01 ng. This system also identified the presence of human DNA in two blood stains stored for 20 and 38 years. The results suggested the potential usefulness of FOXP2 as an identifier of human DNA in forensic samples.     

Validation of the AMPFℓSTR® MiniFilerTM PCR Amplification Kit for Use in Forensic Casework*

Abstract: The AmpF?STR^® MiniFiler^TM PCR Amplification Kit is designed to genotype degraded and/or inhibited DNA samples when the AmpF?STR^® Identifiler^TM PCR Amplification Kit is incapable of generating a complete genetic profile. Validation experiments, following the SWGDAM guidelines, were designed to evaluate the performance of MiniFiler. Data obtained demonstrated that MiniFiler, when used in conjunction with Identifiler, provided an increased ability to obtain genetic profiles from challenged samples. The optimum template range was found to be between 0.2 and 0.6 ng, with 0.3 ng yielding the best results. Full concordance was achieved between the MiniFiler kit and Identifiler kit except in a single case of a null allele at locus D21S11. Numerous instances of severe heterozygous peak imbalance (<50%) were observed in single source samples amplified within the optimum range of input DNA suggesting that caution be taken when attempting to deduce component genotypes in a mixture.     

实时RT-PCR检测大鼠死后管家基因mRNA的时序性降解

目的研究实时荧光定量RT—PCR方法检测死亡大鼠管家基因mRNA时序性降解的可行性,为死亡时间（Dostmortem interval,PMI）推断寻找新的研究手段。方法应用SYBR Green Ⅰ实时荧光定量RT—PCR技术．检测死后不同时间大鼠脑和脾中管家基因GAPDHmRNA及β—actinmRNA的水平,结果用循环阈值（简称Ct值）表示,分析死后经过时间与Ct值的线性关系,并建立死亡时间推断回归方程。结果GAPDH mRNA和β—actinmRNA的Ct值均与PMI之间存在显著的相关性。结论SYBR GreenⅠ实时荧光定量RT—PCR在定量分析mRNA降解的研究中是一个较理想的技术手段。选用管家基因作为PMI推断的研究对象,可在法医检案中消除其他基因因为个体差异带来的误差,更具实用性。Ct值作为动态监测机体死后不同时间点的客观指标．与死后不同时间点的线性关系良好,推断死后经过时间尤其是晚期死亡时间较为理想。     

A real-time multiplex SNP melting assay to discriminate individuals

A method that quickly and inexpensively differentiates crime scene samples from multiple donors would expedite casework analysis by allowing the selection of probative items requiring comprehensive testing. This new method need not be perfectly definitive nor give a complete 13 locus short tandem repeat (STR) profile; it simply must be able to differentiate between most victim and suspect samples. We describe the development of multiplex, single nucleotide polymorphism (SNP), fluorescence resonance energy transfer-based real-time polymerase chain reaction (PCR) assays to fulfill this need. Dual probes, one fluorescently labeled and the other labeled with a quencher, are monitored during a melt analysis to reveal an increase in fluorescence, which allows the assessment of the two SNP alleles. Two alternate 6-plex assays (with and without gender determination) have been developed for the six-color RG6000 real-time instrument (Corbett Robotics, Inc.) and one seven SNP plus gender assay (performed as two 4-plex assays, one with gender the other without) have been developed for use in four/five color real-time instruments. This technique can discriminate between 95% and 99% of samples from different individuals. This assay is fast (approximately 2 h), much less expensive than STR analysis, and uses a real-time PCR instrument which is found in most forensic and molecular biology labs.     

大鼠急性心肌缺血后肌浆网RyR2 mRNA表达变化

目的 研究大鼠急性心肌缺血后心肌肌浆网兰尼碱受体蛋白2(ryanodine receptor 2,RyR2)mRNA表达的变化.方法 将SD大鼠分为正常对照组、心肌缺血组和缺血性猝死组.采用腹腔注射垂体后叶素的方法复制大鼠急性心肌缺血和猝死模型,对心肌进行半定量荧光RT-PCR检测,观察RyR2 mRNA表达水平的变化.结果 与正常对照组相比,不同时间和不同程度的急性心肌缺血后心肌肌浆网RyR2 mRNA表达均显著降低(P<0.05).结论 心肌缺血性损伤可诱导心肌钙调控蛋白RyR2 mRNA表达下调.