Development of Rapid and Economical Colorimetric Screening Method for p‐Phenylenediamine in Variety of Biological Matrices and its Application to Eleven Fatal Cases of p‐Phenylenediamine Poisoning

A rapid colorimetric method for detection of p‐phenylenediamine (PPD) in various biological samples is developed. The o‐cresol test for acetaminophen detection has been modified to detect PPD in blood, urine, gastric contents, and liver. After precipitating protein with trichloroacetic acid solution (2 mL, 10% w/v), biological specimens were required to convert PPD metabolites to PPD by acid hydrolysis. Finally, o‐cresol solution (1 mL, 1% w/v), hydrogen peroxide (200 μL, 3%v/v), and concentrated ammonium hydroxide (0.5 mL) were added in the biological samples. The presence of PPD was indicated by formation of violet color which was turned to bluish green color within 10–15 min. The limit of detection was found to be 2 mg/L in blood, urine, and gastric contents and 2 mg/Kg in liver. This method is also free from any potential interference by p‐aminophenol, acetaminophen, and other amine drugs under test conditions. This method was successfully employed to thirteen fatal cases of PPD poisoning.     

The Discrimination of Colored Acrylic,Cotton, and Wool Textile Fibers Using Micro‐Raman Spectroscopy. Part 1: In situ Detection and Characterization of Dyes

Raman spectroscopy has been applied to characterize fiber dyes and determine the discriminating ability of the method. Black, blue, and red acrylic, cotton, and wool samples were analyzed. Four excitation sources were used to obtain complementary responses in the case of fluorescent samples. Fibers that did not provide informative spectra using a given laser were usually detected using another wavelength. For any colored acrylic, the 633‐nm laser did not provide Raman information. The 514‐nm laser provided the highest discrimination for blue and black cotton, but half of the blue cottons produced noninformative spectra. The 830‐nm laser exhibited the highest discrimination for red cotton. Both visible lasers provided the highest discrimination for black and blue wool, and NIR lasers produced remarkable separation for red and black wool. This study shows that the discriminating ability of Raman spectroscopy depends on the fiber type, color, and the laser wavelength.     

荧光染料掺入PCR检测STR基因座多态性的研究

目的研究荧光染料掺入PCR检测STR基因座多态性。方法 利用荧光染料掺人PCR扩增技术,测定FABP、CYP19、FOLP23、MBP、DYS19五个基因座的梯阶片段长度,将带有荧光标记物的dCTP通过PCR反应加入到目的片段PCR产物中,通过PE 377 DNA测序仪分离和基因扫描软件分析。结果 获得了清晰、准确的图谱,计算出各基因座中核心序列的重复次数,并按照ISFH标准对各基因座命名。结论 该方法具有灵敏度高、结果客观准确、操作简便、产物定量等优点,在新基因座开发和法医鉴定实践中具有较理想的应用价值和发展前景。     

An STR Melt Curve Genotyping Assay for Forensic Analysis Employing an Intercalating Dye Probe FRET*

Abstract: The most common markers used in forensic genetics are short tandem repeats (STRs), the alleles of which are separated and analyzed by length using capillary electrophoresis (CE). In this work, proof of concept of a unique STR genotyping approach has been demonstrated using asymmetric PCR and a fluorescence resonance energy transfer (FRET)‐based hybridization analysis that combines fluorophore‐labeled allele‐specific probes and a DNA intercalating dye (dpFRET) in a melt match/mismatch analysis format. The system was successfully tested against both a simple (TPOX) and a complex (D3S1358) loci, demonstrated a preliminary detection limit of <10 genomic equivalents with no allelic dropout and mixture identification in both laboratory‐generated and clinical samples. With additional development, this approach has the potential to contribute to advancing the use of STR loci for forensic applications and related fields.     

LC—MS／MS法测定蓝色圆珠笔油墨中的碱性染料

目的建立蓝色圆珠笔油墨中碱性染料的LC—MS／MS方法,为蓝色圆珠笔油墨的种类鉴别提供方法。方法用二级质谱寻找并确定结晶紫、甲基紫、维多利亚蓝B、碱性紫14、碱性蓝7和罗丹明B等碱性染料的特征性母离子／子离子对。收集50种蓝色圆珠笔．划线后对其笔道用0．5mm直径打孔器取样,乙腈超声提取。液相分离采用WatersXBridgeC18柱。流动相为0．1％甲酸缓冲液（A）-乙腈（B）,梯度程序洗脱。结果4个点的取样量足以满足检测需要,采用相对峰面积的定量方法．结果重现性好．RSD％≤2．3％。应用该方法对50种蓝色圆珠笔油墨中的碱性染料进行检测,区分率为94．4％。结论所建LC—MS／MS方法定性准确,定量可靠,为蓝色圆珠笔油墨的种类鉴别提供了方法。     

Visualising latent DNA on tapes

This study reports a simple method for visualising and screening latent DNA on tapes using a Diamond™ dye (DD) staining process followed by visualisation using a portable fluorescence microscope. Ten types of tapes were tested, which include those used currently by forensic laboratories for tape-lifting. All ten types were tested for: 1) their auto-fluorescence, 2) properties when stained with DD using three different DD solutions, and 3) PCR inhibition through a direct STR amplification technique. No background fluorescence was noted viewing four types stained with 20 x DD diluted with 0.01% Triton-X. Clear tape (Sellotape®), DNA-free tape (Lovell Surgical Solutions) and brown packing tape (Packmate™) did not inhibit direct STR amplification, while the other six types showed the inhibition of the PCR. The three tapes were selected to assess their cellular material recovery efficiency by comparing the number of stained cells within an entire fingermark before and after tape-lifting. Tape-lifting was performed either once, twice or ten times. The DNA-free tape (Lovell) used in many forensic laboratories gave poor recovery compared to the clear tape (Sellotape®) and brown packing tape (Packmate™). This simple visualising technique allows the cell location to be recorded, and only the area of tape where cells are present to be removed for DNA typing. The process is a simple and effective triage procedure that reduces the processing of tape-lift samples where there are no cells present.     

利用分散染料对血手印染色的研究

本文着重就分散染料对非渗透性客体上血手印的显现方法进行了科学详细的阐述,且突破原有传统的血手印显现法,开辟出用分散染料显现血手印的新途径,在大量实验的基础上,用各种分散染料以铝合金、PVC板、矽钢片、油漆木、玻璃等非渗透性客体上遗留的血手印为主要实验对象,对血手印包括血潜手印进行显现。通过在长波紫外灯下观察比较效果,最终发现荧光黄Ⅱ分散染料显现效果最佳,其显现的血手印荧光强度高,显出的纹线清晰、连贯性好,尤其加入适量的媒染剂后,手印与背景的反差更大,效果更加明显,是一种可靠的手印显现用染料。     

High-performance liquid chromatography-ultraviolet-visible spectroscopy-electrospray ionization mass spectrometry method for acrylic and polyester forensic fiber dye analysis

A critical point of comparison between a fiber collected from a crime scene and a fiber from a known source is the color. Fiber dye analysis using thin-layer chromatography or ultraviolet (UV)-visible (Vis) microspectrophotometry provides useful, although limited, data for comparison. High-performance liquid chromatography-electrospray ionization mass spectrometry (LC/MS) overcomes these limitations by integrating chromatography, ultraviolet-visible spectroscopy, and mass spectrometry into a single instrument. In order to evaluate the applicability of the LC/MS to forensic fiber dye analysis, a multi-stage chromatographic method using acidified water and acidified acetonitrile was developed that separated and identified a mixture of 15 basic and 13 disperse dye standards. The LC/MS also detected and analyzed dyes extracted from individual 0.5 cm acrylic and polyester fibers, demonstrating its applicability to this type of analysis. With regard to the analysis of disperse dyes in polyester fibers, the replacement of pyridine with acetonitrile in the extraction system allowed direct injection of the extracts into the LC/MS. The advantage of the LC/MS over other instrumental methods of textile dye analysis is demonstrated by the analysis and differentiation of three black acrylic fibers: two fibers had similar UV-Vis spectra but were differentiated with chromatography and two had similar UV-Vis spectra and chromatograms but were differentiated using the mass spectrometer.     

Increased sensitivity for amplified STR alleles on capillary sequencers with BigDye XTerminator™

Free fluorescent dyes from PCR primers or amplification products can interfere with the interpretation of STR alleles in an electropherograph especially when the profiles have a low signal intensity. These artefacts can be removed by using a simple procedure based on BigDye^® XTerminator™. This procedure requires limited amounts of PCR product, allows to do several loadings on a capillary sequencer starting from the same purified PCR product and also increases the sensitivity for detection of less amplified loci.     

Detection of cellular material within handprints

A novel technique for the visualisation of cellular material has been published harnessing an external binding nucleic acid fluorescence dye, Diamond™ dye (DD), in combination with a digital fluorescence microscope. This technique can effectively detect cellular material on an object transferred by touch allowing targeted collection of latent DNA. Previous studies on the visualisation of touch DNA have focussed on transfer from fingertips only.Here we report on the visualisation of cellular material transferred via twenty different positions over the entire handprint. Three volunteers (a heavy, an intermediate and a light shedder) were asked to press their hands onto a plastic surface with medium pressure for 15 s at undefined time points post-handwashing, creating a complete handprint. DD was applied to the entire area and the presence of cellular material was recorded based on cells within 5 separate frames at each of the 20 positions. All tests were performed in triplicate such that the final dataset contained 1,800 observed frames.This extensive study allows accurate monitoring of cellular transfer deposited by different parts of the hand. Our study highlights which areas of an individual’s hand shed the greatest, or least, amount of cellular material. This simple process can act as a guide for DNA collection from items held within the entire hand, rather than only touched by the fingertips only, such as weapons, knives and steering wheels.