浓缩DNA法结合miniSTR分型技术检验微量DNA

目的优化低拷贝数DNA STR分型方法。方法对采用磁珠法或Chelex-100法提取DNA,Identi-filer试剂盒扩增,未获得分型结果的日常检案检材,采用物理浓缩法或过柱浓缩法浓缩DNA,采用miniFilerTM试剂盒再次扩增分型。结果 127例检材中,47例磁珠法提取DNA未获得分型的样品,分型成功率为36%;80例Chelex-100法提取DNA未获分型的样品,分型成功率为30%。结论采用浓缩法和miniFilerTM试剂盒,可以提高日常检案中低拷贝数检材的STR检验分型成功率。     

常见嗜尸性甲虫及蝇类虫体不同部位mtDNA的提取效果

目的探讨改良后十六烷基三甲基溴化铵(cetyltriethylammnonium bromide,CTAB)法对常见嗜尸性昆虫虫体不同部位mtDNA的提取效果。方法随机采集放置在呼和浩特地区室外草地家兔尸体上丽蝇科的丝光绿蝇[Lucilia sericata(Meigen)]、埋葬甲科的Nicrophorus fossor(Erichson)各13例,采用改良CTAB法分别提取每只昆虫头部、胸肌、腿部、翅膀4个部位的mtDNA,核酸蛋白测定仪检测DNA纯度及质量浓度,琼脂糖凝胶电泳检测其PCR产物,测定PCR产物序列,上传GenBank。结果 13例丝光绿蝇[Lu-cilia sericata(Meigen)]的胸肌均提取到了DNA,10例头部样本、6例腿部样本、4例翅膀样本得到DNA;13例Nicrophorus fossor(Erichson)胸肌DNA均提取成功,5例头部样本、8例腿部样本及3例翅膀样本提取出DNA。结论改良CTAB法可用于对嗜尸性昆虫胸肌及其他部位进行DNA的提取。     

猪传染性胃肠炎病毒S-N融合双基因疫苗的构建及其免疫原性分析

为了构建TGEV S-N融合双基因疫苗并分析其免疫原性,从S、N基因克隆质粒中以PCR扩增了S基因(2.1kb,含A、B、C、D抗原位点)和N基因(1.2kb),将S基因插入pVAX1载体构建了pVAX-S质粒,再将N基因插入pVAX-S中S基因末端,构建了融合表达S、N双基因的重组质粒pVAX-S-N,将pVAX-S-N转染COS7细胞以免疫荧光试验进行S、N双基因的表达鉴定。用纯化的pVAX-S-N和作为对照的pVAX-S、pVAX1、PBS免疫BALB/c小鼠,共免疫3次,分别测定免疫后第0、14、28、42天的小鼠血清IgG抗体,测定免疫后第42天小鼠外周血T淋巴细胞亚群(CD3+、CD4+、CD8+)的数量。结果,融合质粒pVAX-S-N可在COS7细胞特异性表达S、N两个蛋白,pVAX-S-N免疫小鼠后第14天即可诱导产生抗TGEV的特异性IgG,但pVAX-S-N诱导的抗体水平一直低于pVAX-S诱导的抗体水平,在免疫后第42天差异极显著(P<0.01);pVAX-S-N可激发小鼠产生细胞免疫应答,但pVAX-S-N组的CD3+、CD4+、CD8+数量均低于pVAX-S免疫组。研究结果表明,融合双基因疫苗pVAX-S-N具有免疫原性,但免疫效果却不如单基因疫苗pVAX-S的理想。     

Trace DNA Sampling Success from Evidence Items Commonly Encountered in Forensic Casework

Trace DNA analysis is a significant part of a forensic laboratory's workload. Knowing optimal sampling strategies and item success rates for particular item types can assist in evidence selection and examination processes and shorten turnaround times. In this study, forensic short tandem repeat (STR) casework results were reviewed to determine how often STR profiles suitable for comparison were obtained from “handler” and “wearer” areas of 764 items commonly submitted for examination. One hundred and fifty‐five (155) items obtained from volunteers were also sampled. Items were analyzed for best sampling location and strategy. For casework items, headwear and gloves provided the highest success rates. Experimentally, eyeglasses and earphones, T‐shirts, fabric gloves and watches provided the highest success rates. Eyeglasses and latex gloves provided optimal results if the entire surfaces were swabbed. In general, at least 10%, and up to 88% of all trace DNA analyses resulted in suitable STR profiles for comparison.     

Evaluation of a Freezer Mill for Bone Pulverization prior to DNA Extraction: An Improved Workflow for STR Analysis

Traditional methods for bone pulverization typically generate heat, risking stability of DNA sample. SPEX? has developed cryogenic grinders which introduce liquid nitrogen to cool the sample and aid in the grinding process. In this study, the Freezer Mill 6970 EFM was used with two DNA extraction methods and routine downstream STR analysis procedures. DNA from as little as 0.1 g of bone powder was used to develop full STR profiles after freezer mill pulverization, and the method was reproducible. Further, no contamination was detected upon cleaning/reuse of the sample vials. There were no significant differences in DNA yield, STR alleles detected, or peak heights using the freezer mill as compared to traditional grinding, and successful DNA profiles were achieved from as low as 0.1 g of bone powder with this method. Overall, this work indicates that this cryogenic mill method may be used as a viable alternative to traditional tissue grinders.     

Comparison between Temperature Gradient Gel Electrophoresis of Bacterial 16S rDNA and Diatom Test for Diagnosis of Drowning

When a body is discovered in water, it is difficult to conclude whether the cause of death was drowning, even today. Although diatom testing by the digestive method is classical, we hypothesized that aquatic bacteria, as well as diatoms, might be detected in drowned bodies, and conducted temperature gradient gel electrophoresis (TGGE)‐targeting 16S rDNA. DNA was extracted from the site water, and from heart blood and liver samples from 27 bodies concluded as drowning deaths by autopsy and subjected to TGGE after amplification of 16S rDNA by polymerase chain reaction. We observed whether the feature point of each 16S rDNA from the site water and blood or liver samples matched. Considerably higher correspondence was observed in drowned bodies, and the rate was higher than that achieved with the digestive method. Moreover, TGGE is safer than the digestive method. Our study suggests that this method can aid diagnosis of drowning.     

Evaluation of Skin Surface as an Alternative Source of Reference DNA Samples: A Pilot Study

An acceptable area for collecting DNA reference sample is a part of the forensic DNA analysis development. The aim of this study was to evaluate skin surface cells (SSC) as an alternate source of reference DNA sample. From each volunteer (n = 10), six samples from skin surface areas (forearm and fingertips) and two traditional samples (blood and buccal cells) were collected. Genomic DNA was extracted and quantified then genotyped using standard techniques. The highest DNA concentration of SSC samples was collected using the tape/forearm method of collection (2.1 ng/μL). Cotton swabs moistened with ethanol yielded higher quantities of DNA than swabs moistened with salicylic acid, and it gave the highest percentage of full STR profiles (97%). This study supports the use of SSC as a noninvasive sampling technique and as a extremely useful source of DNA reference samples among certain cultures where the use of buccal swabs can be considered socially unacceptable.     

Forensic Use of the Piracatinga Fish (Callophysius macropterus) to Locate and Identify Human Remains Retrieved From the Amazon River

Piracatinga (Callophysius macropterus) are a type of bottom feeder catfish encountered in the Amazon River and its tributaries. We report two cases in which human remains were first located based on a characteristic circular distortion of the surface of the river that the Piracatinga make while they feed. Human skin samples of one of the victims recovered from the Piracatinga digestive tract were subjected to mitochondrial DNA analysis that allowed identification of the body of Case 1; the family recognized body parts of Case 2. Importantly, the location of human body parts and their identification based on DNA analysis enabled the respective families to obtain a death certificate expeditiously in the absence of identifiable remains—a process that normally requires 5 years under current Brazilian law, and in the absence of closure, imposes severe emotional stress on the family of the deceased.     

The effect of mark enhancement techniques on the presumptive and confirmatory tests for blood

An investigation into the effects of physical and chemical enhancement on subsequent presumptive and confirmatory tests for human blood is presented. Human blood was deposited onto porous (white 80 gsm paper and brown envelope) and non-porous (tile and linoleum) substrates in a depletion series (30 depletions on non-porous and 20 on porous) and subjected to three ageing periods; 1, 7 and 28?days. A number of enhancement techniques were tested [fluorescence, black magnetic powder (BMP), iron-oxide black powder suspension (PS), cyanoacrylate (CA) fuming, acid violet 17 (AV17), acid yellow 7 (AY7), ninhydrin, DFO and Bluestar Forensic Magnum (BFM) luminol] to evaluate their potential effects on subsequent presumptive and confirmatory tests. AV17 and Bluestar provided the best enhancement and fully enhanced all depletions in the series. The sensitivity of the Kastle-Meyer (KM) (presumptive), Takayama and RSID-Blood tests (confirmatory) was initially investigated to determine the range of detectable depletions. The KM test detected all depletions, whereas the Takayama test detected up to depletion 6 and RSID-Blood detected up to depletion 20 (paper), 10 (envelope), 15 (tile) and 9 (lino). The abilities of these tests to detect blood after enhancement were then observed.A number of techniques resulted in little to no effect on any of the blood tests, whereas adverse effects were observed for others. Ninhydrin and CA fuming caused weak but instantaneous positive KM results whereas methanol-based AV17 and AY7 delayed the reaction by as much as 1?min. The Takayama test was not very sensitive, therefore, its performance was easily affected by enhancement and negative results were often observed. RSID-Blood tests were largely unaffected by chemical enhancement although a drop in positive results was observed for some of the techniques when compared to positive controls.Using a standard procedure for DNA extraction, all the tested blood samples (before and after enhancement) gave a detectable quantity of DNA and were successfully profiled. Out of the 45 samples processed for DNA profiling, 41 gave full profiles, while the remaining showed allele drop out in one or two loci.     

Single source DNA profile recovery from single cells isolated from skin and fabric from touch DNA mixtures in mock physical assaults

The ability to obtain DNA profiles from trace biological evidence is routinely demonstrated with so-called ‘touch DNA evidence’, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from a donor's hands to an object or person during direct physical contact. Current methods for the recovery of trace DNA employ swabs or adhesive tape to sample an area of interest. While of practical utility, such ‘blind-swabbing’ approaches will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even though the individuals' cells are principally located in topographically dispersed, but distinct, locations on the item. Thus the act of swabbing itself artifactually creates some of the DNA mixtures encountered in touch DNA samples. In some instances involving transient contact between an assailant and victim, the victim's DNA may be found in such significant excess as to preclude the detection and typing of the perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods for touch DNA evidence, we reported previously the development of a ‘smart analysis’ single cell recovery and DNA analysis method that results in enhanced genetic analysis of touch DNA evidence. Here we use the smart single cell analysis method to recover probative single source profiles from individual and agglomerated cells from various touched objects and clothing items belonging to known donors. We then use the same approach for the detection of single source male donor DNA in simulated physical contact/assault mixture samples (i.e. male ‘assailant’ grabbing the wrist, neck or clothing from the female ‘victim’, or being in transient contact with bedding from the ‘victim’). DNA profiles attributable to the male or female known donors were obtained from 31% and 35% of the single and agglomerated bio-particles (putative cells) tested. The known male donor ‘assailant’ DNA profile was identified in the cell sampling from every mixture type tested. The results of this work demonstrate the efficacy of an alternative strategy to recover single source perpetrator DNA profiles in physical contact/assault cases involving trace perpetrator/victim cellular admixtures.