大鼠死后脑、心肌和肾组织β-actin mRNA的降解与早期死亡时间的关系

目的 研究SD大鼠脑、心肌和肾组织细胞内β-actin mRNA的降解与早期死亡时间的关系,为早期死亡时间的推断寻找新的指标.方法 大鼠处死后置于20℃的环境中,分别于死后不同时间点提取脑、心肌、肾的总RNA,采用实时荧光定量RT-PCR方法检测总RNA中β-actin mRNA的水平(Ct值),分析死后经过时间与Ct...     

Analysis of Dopamine D2 Receptor (DRD2) Gene Polymorphisms in Cannabinoid Addicts*

Abstract: The gene encoding the dopamine D2 receptor (DRD2) has been suggested as a candidate gene for substance dependence. In this study, the possible association between Taq1A and Taq1B DRD2 polymorphisms and cannabinoid dependence was investigated. One hundred and twelve cannabinoid addicted and 130 healthy control subjects were included in this study. The Taq1A and Taq1B genotypes were determined in all subjects by polymerase chain reaction. For each polymorphism (A or B), the subjects were categorized into three groups according to their genotype, that is, the subjects with alleles A1/A1, A1/A2, A2/A2; B1/B1, B1/B2, and B2/B2. A significant association was found between Taq1A gene polymorphism and cannabinoid addicts compared to the control subjects. This finding suggests that polymorphism of the Taq1A, but not the Taq1B, may be associated with the susceptibility to cannabinoid dependence. Further clinical studies are required to be carried out for confirmation and evaluation of these findings.     

应用RNA分析技术推断现场血痕形成时间

目的检测血痕中β-actin mRNA和18S rRNA在人体死后8～15d期间的表达残留,为推断血痕形成时间寻找新的客观依据。方法在上述时段内每天抽提血痕总RNA,利用实时定量RT-PCR技术监测18S rRNA和β-actin mRNA的扩增状况并对产物进行定量分析,通过检测18S rRNA与β-actin mRNA含量在各个时间点的变化趋势来推测血痕形成时间。结果死后8～15 d时段内18S rRNA与β-actinmRNA量的比值呈明显升高趋势,显示出两种不同类型RNA降解的时间差异性。结论一定时段内18SrRNA与β-actin mRNA量的相对变化,可作为推测血痕形成时间的参考指标。     

Molecular Identification of Three Indian Snake Species Using Simple PCR–RFLP Method*

Abstract: Three endangered Indian snake species, Python molurus, Naja naja, and Xenochrophis piscator are known to be significantly involved in illegal trade. Effective authentication of species is required to curb this illegal trade. In the absence of morphological features, molecular identification techniques hold promise to address the issue of species identification. We present an effective PCR–restriction fragment length polymorphism method for easy identification of the three endangered snake species, Python molurus, Naja naja, and Xenochrophis piscator. A 431‐bp amplicon from cytochrome b gene was amplified using novel snake‐specific primers following restriction digestion with enzymes Mbo II and Fok I. The species‐specific reference fragment patterns were obtained for the target species, which enabled successful identification of even highly degraded shed skin sample confirming the utility of the technique in case of poor‐quality DNA. The assay could be effectively used for forensic authentication of three Indian snake species and would help strengthen conservation efforts.     

Simultaneous Identification of Four “Legal High” Plant Species in a Multiplex PCR High‐Resolution Melt Assay,

The international prevalence of “legal high” drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real‐time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high‐resolution melt using LCGreen Plus^®. The PCR assay was evaluated based on the following: (i) specificity and selectivity—primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity—serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability—sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging “legal high” species.     

Development of a PCR High‐Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional “Unregulated Legal High” Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose),

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone‐free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high‐resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.     

温州汉族人群D7S2846、D19S400和D18S535基因座的遗传多态性研究

目的研究D7S2846、D19S400和D18S535位点在温州汉族人群的遗传多态性。方法EDTA抗凝血样采自194名无血缘关系温州地区汉族个体,用chelex-100法提取DNA,PCR扩增,聚丙烯酰胺凝胶电泳,银染显色分析。结果D7S2846观察到6个等位基因及15种基因型;D19S400观察到10个等位基因及36种基因型;D18S535观察到8个等位基因及26种基因型。各基因座的杂合度(H)分别为:0.644、0.724、0.772;个人识别能力(Dp):0.854、0.940、0.938。结论三个STR基因座具有较高杂合度,等位基因分布符合Hardy-Weinberg平衡,在法医学应用和群体遗传学研究中有较高的价值。     

Use of a commercially available hydrogel as a novel DNA surface sampling tool

A common requirement in the military, law enforcement, and forensic mission space is the need to collect trace samples from surfaces using a method that not only readily captures the sample but also retains its integrity for downstream identification and characterization. Additionally, collecting samples from three-dimensional objects (e.g., shell casings) is a challenge for which there is currently no validated standardized approach. Recently, hydrogels have been shown to have the potential for surface collection of trace bacterial spores, amino acids, and DNA. To test whether these hydrogels can serve as a viable collection medium for sampling DNA from surfaces, we carried out a series of preliminary tests examining collection efficiency and suitability of hydrogel material to recover samples of diluted, dried human DNA on a smooth polycarbonate surface. The recovery of surface DNA using a commercially available hydrogel was examined, and the efficiency compared to samples collected using a standard foam collection swab. DNA collected using the hydrogel and swab methods was then examined using quantitative polymerase chain reaction (qPCR) and short tandem repeat (STR) analysis to determine whether the collection material was compatible with these downstream processes. The hydrogel material used for this study collected the experimental DNA with comparable efficiency to standard collection swabs. In addition, qPCR and STR analyses demonstrated compatibility with the hydrogel collection and extraction process. These data suggest that hydrogels have the potential to be used as sample collection materials and deserve further characterization to elucidate their utility in collection from irregularly shaped, three-dimensional surfaces/materials.     

A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology

Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single-cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30–150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well-performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%–75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 μL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single-cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.     

15重快速STR复合扩增体系的构建

目的建立一套15重快速STR复合扩增体系。方法选择14个常染色体基因座以及1个性别基因座,采用Fast Start Taq DNA聚合酶系统,以DNA标准品9947A为模板,通过筛选扩增条件、选择热启动酶用量、调整引物平衡、优化快速扩增程序、筛选反应缓冲液、选择反应体系以及筛选添加剂等一系列复合扩增实验,比较各条件下等位基因丢失和非特异性扩增情况。结果在以1 ng DNA为模板、0.4μL聚合酶及10×Fast Start高保真反应缓冲液构成10μL快速体系的条件下,32 min即可获得标准DNA全部15个STR基因座的完整分型,无等位基因丢失和非特异性扩增现象,等位基因均衡性良好。同时,5%甘油、0.01%明胶、0.05%明胶和5 mmol/L硫酸铵可作为PCR扩增过程中拟加入的反应添加剂。结论本研究建立的15重快速STR复合扩增体系可以明显缩短反应时间,提高样品检测效率。