差异提取试剂盒在混合斑DNA提取中的应用

目的评估差异提取试剂盒对混合斑样本中的精子和上皮细胞DNA分离提取的有效性。方法采用差异提取试剂盒,选择性裂解精细胞和上皮细胞,结合磁珠法分别对人为控制条件下制备的模拟混合样本和案件中的混合斑检材进行精细胞DNA和上皮细胞DNA的分离提取。对所提取的DNA进行定量分析和STR分型。结果该试剂盒能从精子和上皮细胞不同比例的混合斑中提取出高纯度的精细胞和上皮细胞DNA。结论该差异提取试剂盒适用于性侵害案件中混合斑检材的DNA提取。     

快速溶剂萃取-凝胶色谱净化-LC／MS／MS相结合测定血中的杀参毒素类农药

目的建立血中杀参毒素类农药残留的快速分析方法。方法取一定量的Al2O3放入萃取池中做吸附剂,通过快速溶剂萃取仪萃取血液中杀参毒素类农药,同时达到在线净化的效果,后运用凝胶色谱净化联用仪进一步净化浓缩到2ml,提取液经LC／MS／MS进行检验。结果杀虫双在1．2×10g／ml～1．2×10^-6g／ml范围内线性关系良好,检出限为0．04ppb,杀虫眯为1．2×10^-6g／ml～7．2×10g／ml,检出限为0．34ppm。2种杀参毒素农药的平均回收率为89％、90％,相对标准偏差为1．9％、3％。结论整个方法简便、快速、准确、重现性好、灵敏度高,杂质干扰少。     

Automated extraction of DNA and PCR setup using a Tecan Freedom EVO liquid handler

We have implemented and validated automated methods for DNA extraction and PCR setup developed for a Tecan Freedom EVO^® liquid handler mounted with a Te-MagS™ magnetic separation device. The DNA was extracted using the Qiagen MagAttract^® DNA Mini M48 kit. The DNA was amplified using AmpF?STR^® Identifiler^®, Y-filer^® (Applied Biosystems), GenePrint^® FFFL and PowerPlex^® Y (Promega). The methods were validated for fresh whole blood and blood from deceased according to EN/ISO 17025.     

Genotyping of DNA samples under adverse conditions of Low Copy Number—LCN (formolisados tissue samples and embedded in paraffin)

This paper aims to describe and evaluate a protocol for extraction of DNA (deoxyribonucleic acid) in formalinized tissues and embedded in paraffin for forensics genetic analysis. In outline the method is the removal of paraffin with an organic solvent in 0.3–0.5 mg of the sample of the tissue under study, followed by removal of formaldehyde, rehydration and soon after the extraction of genomic DNA. The extraction is achieved through the stages of cellular lysis, enzymatic digestion of proteins and DNA precipitation in ethanol medium. With the research we can conclude that even when the DNA is present in small quantities in conditions of extreme difficulties in its extraction, as formalinized tissues and embedded in paraffin, the technique of optimizing the extraction of DNA used both to organic extraction as Chelex, for use in the polymerase chain reaction (PCR), and possible the investigation of different samples of human tissue, biological samples, or was obtained under the conditions tested, a DNA with good quality and concentration. The samples were amplified for the mini-STRs loci using the product marketed in multilocus, using a methodology recommended by the supplier and validated for analysis of forensic DNA. Commercial kit was used MiniFiler from Applied Biosystems. The DNA fragments amplified by PCR showed that the extracted DNA had good amplification.     

A new approach in the identification of degraded paternity samples

DNA extraction from bone is an important issue particularly in paternity cases when bones are the only remaining material to obtain and analyze DNA. The difficulties arising from bacterial damages, taphonomic factors and diagenesis might negatively affect the extraction and the amplification of DNA. This makes the laboratory procedure a hard and time-consuming process, and the analysis can fail. Analyzing mini-STRs in this type of degraded samples is highly recommended. In this study a new extraction technique was carried on bone samples which were then typed for mini-STRs. The aim was to differentiate two genetically related skeletons found in the same familial grave for a paternity test. The analysis revealed that this new extraction technique along with mini-STR analysis can properly be an effective way to obtain and analyze DNA in bones in the field of forensic sciences.     

Evaluation of DNA Extraction Methods for Processing Fingerprint Powder-Coated Forensic Evidence

In unison, fingerprinting and DNA analysis have played a pivotal role in forensic investigations. Fingerprint powders that are available on the market can come in a range of colors and with specific properties. This study evaluated the efficiency of DNA extraction from samples coated with 3 brands of fingerprint powders: Lightning, Sirchie, and SupraNano, covering a range of colors and properties. A total of 23 fingerprint powders were tested using the Chelex, Promega DNA IQ™, and Applied Biosystems™ PrepFiler™ DNA extraction protocols. The DNA IQ™ and PrepFiler™ methods extracted higher yields of DNA in comparison to Chelex, which also accounted for better quality of PowerPlex x00AE; 21 DNA profiles recovered. There were no signs of degradation or inhibition in the quantification data, indicating that samples returning low DNA yield was due to interference during DNA extraction and not PCR inhibition. DNA profiles were recovered from the majority of fingerprint powders with only a single powder, Sirchie Magnetic Silver, failing to produce a profile using any of the methods tested. A link was observed between the DNA extraction chemistry, fingerprint powder property, that is, nonmagnetic, magnetic and aqueous, and the brand of fingerprint powder. Overall, the DNA IQ™ method was favorable for nonmagnetic fingerprint powders, while magnetic fingerprint powders produced more DNA profiles when extracted with the PrepFiler™ chemistry. This study highlights the importance of screening DNA extraction chemistries for the type of fingerprint powder used, as there is not a single DNA extraction method that suits all fingerprint powder brands and properties.     

Comparison of the M-Vac® Wet-Vacuum-Based Collection Method to a Wet-Swabbing Method for DNA Recovery on Diluted Bloodstained Substrates*†‡

A wet-vacuum-based collection method with the M-Vac^® was compared to a wet-swabbing collection method by examining the recovery of diluted blood on 22 substrates of varying porosity. The wet-vacuum method yielded more total nuclear DNA than wet-swabbing on 18 porous substrates, recovering on average 12 times more DNA. However, both methods yielded comparable amounts of total DNA on two porous and two nonporous substrates. In no instance did wet-swabbing significantly recover more DNA. The wet-vacuum method also successfully collected additional DNA on previously swabbed substrates. Mitochondrial DNA yields were assessed, and outcomes were generally similar to the nuclear DNA outcomes described above. Results demonstrate that wet-vacuuming may serve as an alternative collection method to swabbing on difficult porous substrates and could potentially recover additional DNA on previously swabbed substrates. However, swabbing remains the preferred collection method on substrates with visible stains and/or nonporous surfaces for reasons of convenience, simplicity, and lower cost relative to the wet-vacuum method.     

多指标正交试验优选炮苍耳子中酚酸类成分的提取工艺

目的 优选苍耳子中酚酸类成分的最佳提取工艺。方法 以苍耳子浸膏得率、5种酚酸类成分及总酚酸的含量为指标,通过正交试验设计,优选出苍耳子中酚酸类成分的最佳水提取工艺。结果 所优选出的最佳提取工艺为10倍量水,浸泡30 min,提取3次,每次90 min。其中提取次数对提取工艺的影响,差异具有统计学意义（P＜0.05）。结论 所选用的提取工艺稳定、简便、可靠,为进一步提取苍耳子中酚酸类成分提供实验参考。     

Fentanyl transdermal patches: Extraction and evaluation of a novel disposal method using NarcX®

Currently, there is no known commercially available product for disposing of used fentanyl transdermal patches. To eliminate the potential for harm and abuse, a proper disposal method is needed–one that neutralizes the dangerous amount of residual fentanyl that remains after therapeutic use of the fentanyl patch. The patent-pending liquid solution of activated carbon, known as NarcX^®, was investigated as a potential fentanyl adsorbing agent. In order to determine the amount of fentanyl remaining after a patch is treated with NarcX^®, here, we utilized hexanes to first dissolve the patch adhesive and then followed with liquid-liquid extraction with methanol to recover the fentanyl. Using liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS), the extracts obtained with this method yielded between 85% and 117% recovery of fentanyl from new and unused patches. Further optimization of this method allowed for a quantitative evaluation of NarcX^®-treated fentanyl patches. 100 µg/h Apotex brand fentanyl patches were exposed to NarcX^® for 1, 24, 48, and 72 h. NarcX^® was shown to adsorb fentanyl from the patches with varying degrees of success, demonstrating an average of 66.98 ± 0.75% fentanyl adsorption after 72 h. These findings suggest that more work is needed to successfully neutralize the fentanyl patches in their entirety using NarcX^®; however, this work does demonstrate proof of concept.     

快速溶剂萃取-GC／MS法测定全血中沙蚕毒素

目的采用快速溶剂萃取-GC／MS分析方法检验血液中杀虫双代谢产物沙蚕毒素。方法样本用硅藻土、中性三氧化二铝处理，用乙酸乙酯：环己烷：丙酮（2：2：1）进行萃取，GC／MS方法进行测定，并考察实验最佳条件。结果沙蚕毒素在50～2000ng／mL范围内线性关系良好，相关系数为0．999，检测限为15ng／mL，平均回收率为98．22％，相对标准偏差为4．6％。结论速溶剂萃取-GC／MS分析方法简便，快速，准确、灵敏度高，可在血中沙蚕毒素检验中选用。