5种免提取试剂盒检验滤纸血痕结果的比较

目的探讨5种免提取试剂盒对滤纸血痕样本检验的效果。方法陈旧滤纸血痕(存放时间12～14个月)及新鲜滤纸血痕(存放时间小于1个月)各920份,分别随机分为5组。应用AGCU 17+1、Goldeneye 20A、Powerplex16HS、Identifiler Plus、Identifiler Direct 5种免提取试剂盒进行检验,对比各组检验结果。结果陈旧滤纸血痕5种试剂盒的检验成功率为98.91%～100%,各组间无差异(P>0.05);新鲜滤纸血痕,Identifiler Plus和Identifiler Direct试剂盒检验成功率高于AGCU17+1、Goldeneye 20A及Powerplex 16HS试剂盒(P<0.01);将样本做陈旧化处理后再用成功率较低的3种试剂盒进行检验,成功率分别升至100%、99.46%、99.46%;Identifiler Plus试剂盒扩增循环27次效果优于28次。结论本文5种试剂盒均可用于滤纸血痕的直接扩增检验,但使用AGCU17+1、Goldeneye 20A及Powerplex 16HS试剂盒需将新鲜血痕做陈旧化处理;Identifiler Plus试剂盒需将循环次数降为27次。     

SNaPshot技术检测Y-SNP位点O3-M122单倍群的应用

目的采用SNaPshot技术检测案例检材O3-M122位点6个亚群的分型,并调查其在云南3个民族无关男性人群中的频率分布。方法建立5-plex Y-SNPs复合扩增体系,采用SNaPshot Multiplex试剂盒,检测案例检材O3-M122单倍群下O3*-M122、O3a*-M324、O3a3*-P201、O3a3b*-M7、O3a3c*-M134、O3a3c1*-M117共6个Y-SNPs分型,并对上述6个Y-SNPs在云南佤族、白族、傣族共174名无关男性个体中的分布进行调查。结果 17份案例检材均得到良好的分型结果。上述3个民族分别在3个、5个、5个位点上发现多态性,其中佤族O3a3*-P201分布频率最高(0.430 0);白族O3a*-M324频率最高(0.212 3);傣族O3a3c1*-M117频率最高(0.158 2)。结论多重PCR反应及SNaPshot技术的联合应用适用于法医案件检材DNA检测,3个民族多态性数据可为相关应用所参考。     

The impact of deposition area and time on Touch DNA collected from fabric

Touched items at crime scenes are frequently analysed to help link suspects to crimes, for example, Touch DNA is collected from victims’ clothes in cases such as sexual assault, homicide, theft etc. Tape lifting is the preferred collection method of choice for trace DNA from clothes, fabric items and porous surfaces such as paper, therefore this study investigated the impact of deposition area and time on Touch DNA collected from fabric using minitapes. The amount of Touch DNA collected from the fabric was significantly affected by deposition area (p < 0.05), time (p < 0.05) and the interaction between the deposition area and time (p < 0.05), with the quantity of DNA collected decreasing over time. Also, the buttocks area of the trouser compared to the chest area is more prone to friction from an activity like repeatedly sitting on different surfaces which reduces the amount of Touch DNA available. In conclusion, it is more effective to collect trace DNA from victim clothes as soon as possible after the crime is committed.     

Development of a nanoplate-based digital PCR assay for species identification with mixture deconvolution

In crime scenes, not all biological stains are human in origin. Some exhibits can be from pets living on the premises or from animal products used in food consumption. In addition, it could be necessary to test animal carcasses for other forensic purposes. Often such stains can include mixtures involving humans or other species. Thus, identifying and deconvoluting mixtures of species commonly found in and around a household can be crucial in forensic casework. Different molecular techniques have been employed for species identification such as immunoprecipitation, qPCR, and DNA sequencing.In this project, a nanoplate-based digital PCR assay for species identification was developed, targeting Homo sapiens, canine, feline, bovine swine, pisces, and gallus in two multiplexes. An internal positive control was included in the design. The assay is simple, rapid, and can determine a wide variety of different vertebrates from biological exhibits, as well as in mixtures. Because the assay utilizes digital PCR, the procedure shows sensitivity down to a few copies, even in the presence of larger amounts of a major contributor, making the assay particularly useful in mixture deconvolution. Overall, this assay presents the forensic community with a novel application in which digital PCR can provide a sensitive and specific determination of species.     

直接同步扩增5种基因位点

来用直接同步ＰＣＲ扩增５种基因位点及反向打点杂交技术，使扩增产物ＤＮＡ与膜探针条杂交。再通过酶联底物显色反应对ＬＤＬＲ、ＧＹＰＡ、Ｄ７Ｓ８、Ｇｃ和ＨＢＧＧ进行联合分型。该方法特别适用于微量、陈旧或降解的ＤＮＡ样本，其操作简便、迅速，应用在亲子鉴定案例，亲子关系概率高达９９．７５％。该技术在法医学、遗传学及临床诊断中具有重要价值，并将改变ＤＮＡ分型技术难于标准化的局面．     

对虾白斑综合征病毒LAMP检测方法的建立

根据对虾白斑综合征病毒(WSSV)囊膜蛋白VP28的基因序列设计合成4条特异性引物,以pMDT-VP28重组质粒为标准模板,通过优化反应体系和反应条件,建立了WSSV的环介导等温扩增(LAMP)检测方法,测定LAMP对WSSV-DNA的最低检测限,并与巢式PCR进行了比较。结果显示,该LAMP方法的最佳反应温度为65℃,反应时间为60min;LAMP对WSSV的最低检测限为10copies/μL,而巢式PCR为100copies/μL,表明该LAMP方法的敏感性显著高于巢式PCR检测方法。因此,WSSV的LAMP检测方法比PCR更为简便、快速、灵敏,且无需昂贵的变温仪器,更适应水产养殖的现地检测,本研究为WSSV早期感染的快速检测提供了新方法。     

Gene expression during blow fly development: improving the precision of age estimates in forensic entomology

Forensic entomologists use size and developmental stage to estimate blow fly age, and from those, a postmortem interval. Since such estimates are generally accurate but often lack precision, particularly in the older developmental stages, alternative aging methods would be advantageous. Presented here is a means of incorporating developmentally regulated gene expression levels into traditional stage and size data, with a goal of more precisely estimating developmental age of immature Lucilia sericata. Generalized additive models of development showed improved statistical support compared to models that did not include gene expression data, resulting in an increase in estimate precision, especially for postfeeding third instars and pupae. The models were then used to make blind estimates of development for 86 immature L. sericata raised on rat carcasses. Overall, inclusion of gene expression data resulted in increased precision in aging blow flies.     

荧光标记8个 miniSTR 及 Amelogenin 复合扩增体系的建立

目的建立非CODIS系统miniSTR以及Amelogenin基因座的荧光复合扩增体系。方法筛选8个多态性高的非CODIS系统miniSTR基因座（D20S1082、D6s474、D12ATA63、D9S1122、D2S1776、D1S1627、D3$4529、D2S441）,并结合Amelogenin基因座设计荧光标记引物,优化反应条件,建立复合扩增体系。应用该体系对204份广州地区汉族血样,30个家系样本,及30份降解检材进行检测。结果建立的荧光标记8个miniSTR及Amelogenin复合扩增体系分型结果明确,稳定性好,且所有片段长度均少于200bp,提高了降解检材的分型成功率。在广州汉族人群的累积个人识别率为0．99999993,累积非父排除率为0．992287。结论构建的miniSTR荧光复合扩增体系,操作简便,分型准确,重复性好,对降解检材有效,易于在法医实验室推广应用,可对现有试剂盒起补充作用。