鸡IL-18对禽多杀性巴氏杆菌DNA疫苗的免疫佐剂作用

为了探索鸡IL-18在禽多杀性巴氏杆菌H基因DNA疫苗中的免疫佐剂作用,分别用共表达鸡IL-18基因和禽多杀性巴氏杆菌C48-1H基因的DNA疫苗、鸡IL-18真核表达质粒pcDNA3/cIL-18与禽多杀性巴氏杆菌C48-1H基因的DNA疫苗混合物、禽多杀性巴氏杆菌C48-1H基因的DNA疫苗肌肉注射5周龄鸡,首免后每周采取外周血及外周抗凝血,应用ELISA和MTT法分别检测免疫鸡的体液免疫及细胞免疫水平。二免后第2周用10 LD50禽多杀性巴氏杆菌强毒菌株C48-1进行攻击。结果鸡IL-18能够明显增强禽多杀性巴氏杆菌H基因DNA疫苗的免疫原性,显著提高免疫鸡的体液免疫和细胞免疫水平,并且鸡IL-18与禽多杀性巴氏杆菌H基因共表达时的免疫佐剂作用最强,能强有力地抵抗强毒菌株C48-1的致死性攻击。结果表明,鸡IL-18可作为DNA疫苗的一种理想的免疫佐剂。     

指甲末端微量物证的DNA检验

1材料与方法1.1材料检材取自近两年35例刑事案件,其中受害人指甲31例(男11例,女20例),嫌疑人指甲4例(均为男性)。1.2方法用0.5cm×0.5cm滤纸两步擦拭法[1]转移指甲末端微量物证,加入5%Chelex-100和蛋白酶K,震荡混匀后置56℃保温1h,99℃8min,提取的DNA用Microcon-100柱纯化处理。用Amp FlSTR(ProfilerPlusTM系统复合扩增试剂盒进行STR基因座复合扩增,扩增产物经ABI 3100或ABI 3130X1型基因分析仪荧光检测分型。2结果与讨论在送检的35例检材中,检出含有他人DNA的案例15例,其中受害人为女性的13例,其中婴幼儿1例;男性2例。具体见表…     

Chelex-100提取生物检材DNA实时PCR定量研究

目的研究Chelex-100法提取的生物检材DNA用量与复合STR分型成功率的关系。方法113份各种生物检材采用Chelex-100法提取DNA，应用Quantifiler人类DNA定量试剂盒在ABI 7500荧光定量PCR仪上进行实时PCR定量，同时用Identifiler复合扩增系统在ABI 3100遗传分析仪上对这些DNA样品进行STR分型。结果各种生物检材提取的DNA浓度分别为：37份滤纸、纱布血痕0．042～5．28ng／μl，16份口腔拭子1．15—4．21ng／μl，18份烟头0．016～1．46ng／μl，10份肋软骨0．531—14．40ng／μl，8份肌肉5．75—24．80ng／μl，7份指甲0．788—11．50ng／μl，17份精斑0．79～99．50ng／μl。在建立的8μl扩增体系中，根据上述结果，调整用于复合STR扩增的DNA模板量在0．5—3ng之间，大部分样品可获得完全的STR分型。结论Chelex-100法提取的检材DNA模板用量在0．5—3ng之间可得到有效STR扩增，浓度为0．5ng／μl以上的DNA样品，用小体积模板（1μl）比大体积（3μl）模板扩增效果好。     

Development of STR profiles from firearms and fired cartridge cases

Fired cartridge cases are a common type of evidence found at crime scenes. However, due to the high chamber temperatures and touch nature of this evidence, DNA testing is not commonly sought because it is believed DNA is only present in low levels, whether it is due to initial low levels of DNA and/or DNA degradation from the heat or inhibition of the PCR reaction. Moreover, very few laboratories report STR typing success with fired cases. This study focused on obtaining STR profiles from fired cartridge cases using the AmpFℓSTR^® MiniFiler™ kit, which is designed to amplify DNA from low level, inhibited, and degraded samples. Comparisons to other STR amplification kits were also conducted. In attempt to simulate casework, random individuals loaded cartridges into a firearm. DNA was recovered from the fired cartridge cases using the double swab technique and extracted using an automated large volume DNA IQ™ method. Initially, testing focused on known shedders handling cartridges for 30 s prior to firing. A significantly greater number of alleles was obtained following amplification with the MiniFiler™ kit versus the PowerPlex^® 16 BIO kit. No alleles were observed using the Identifiler^® kit. In an attempt to better simulate casework, a random selection of laboratory personnel handled shotshells for as long as needed to load and fire the weapon. In this mock sample study, the MiniFiler™ kit successfully amplified an average of 22% of expected alleles from DNA recovered from shotshell cases versus the PowerPlex^® 16 BIO kit where an average of 7% of alleles were observed. However, the total number of alleles obtained from the two kits was not significantly different. The quality of the DNA obtained from fired cases was studied with evidence of inhibition in at least 11% of shotshell case samples. After swabbing the head and the hull of three shotshell cases separately, a significantly greater number of alleles was obtained from the hull as opposed to the head of the fired shotshell case. In addition, after firing, various internal firearm surfaces were swabbed, including the chamber of barrel, ejection port, and breechface, in an attempt to obtain amplifiable DNA. DNA was obtained from the chamber of the barrel and was amplifiable using the MiniFiler™ kit, although mixtures were obtained with extensive drop-in and drop-out making this analysis unlikely to aid an investigation.     

印记基因KCNQ1的遗传多态性及在亲权鉴定中的应用

目的为了调查印记基因KCNQ1的STR位点在中国汉族人群中的遗传多态性，利用亲源印记等位基因（parentally imprinting allele，PIA）分型法确定孩子的等位基因亲代来源，为亲权鉴定提供新的侯选STR位点。方法应用Chelex法提取153例佳木斯地区汉族健康无血缘关系个体DNA，用QIAamp Blood Kit（Qiagen）法提取3个家庭10个个体DNA，PCR扩增，凝胶电泳分型，ABIPRISM^TM 3730XL DNA测序仪测序；甲基化敏感性限制性内切酶消化孩子基因组DNA，PCR扩增，确定孩子等位基因的亲代来源。结果发现在中国佳木斯地区汉族人群中KCNQ1基因的STR有7个等位基因，多态信息含量为0．662，且KCNQ1基因的STR位点呈父源印记。结论印记基因KCNQ1的STR位点有很好的多态性，可为亲权鉴定提供新的侯选遗传标记，其亲源特异性甲基化标记有望应用于单亲鉴定中。     

线粒体16SrRNA和Cytb基因复合扩增进行种属鉴定

目的建立一种用于种属鉴定的线粒体DNA16SrRNA基因和细胞色素b基因荧光标记复合扩增检测体系。方法利用引物设计软件Primer5.0对mtDNA序列的16SrRNA基因和细胞色素b基因各设计一对引物,建立复合扩增体系,分别扩增人和牛、猪、狗、鸡、草鱼5种常见动物,用310遗传分析仪对产物进行分析。结果人和5种动物DNA扩增产物均出现两个峰,Cytb通用引物的扩增产物为人与动物的共有峰,为358bp;16SrRNA基因的扩增产物为人与动物间存在位置差异的特异峰,位于231～256bp之间。结论该复合扩增体系可以明确区分人和5种动物样本,可用于种属鉴定。     

刑事案件DNA检验采样与鉴定立法现状

人类DNA遗传特征多态性使DNA技术成为各国警方侦办案件的重要技术手段，但DNA检验鉴定采样合法性和结论可采性须由立法规定。众多国家和地区针对DNA采样和鉴定结论应用制定了相关法律法规。我国相关法律细则尚处空白，因此，应尽快制定我国DNA鉴定采样与应用法规，规范DNA鉴定样本采集与证据采信。     

Evaluation of the use of freezer mill to improve DNA retrieval from dried cotton swabs

This investigation evaluated the use of a freezer mill to improve retrieval of DNA from dried cotton swabs (using 100 μl saliva) compared to uncrushed swabs and whole saliva by measuring DNA yield and profile average peak height. Three treatments were tested; short, medium (as for a bone sample) and extended. The samples subjected to the freezer mill had the powder that remained on the freezer mill components collected (using a water and agitation method). All freezer mill samples returned a lower average DNA yield than either uncrushed swabs or whole saliva. The powder from the crushed swabs comprised an average of 35% of the total DNA yield, whereas the powder from the components comprised 65%. Allele drop-out was observed in samples exposed to extended treatments. Both short and medium treatments provided significantly higher peak heights than uncrushed cotton swabs with equal quantities of DNA (P < 0.05). Using a freezer mill on dried cotton swabs does not increase the DNA yield. This investigation suggests collecting powder from the freezer mill components will increase DNA yield, especially from trace samples.     

中国青海藏族、汉族mtDNA控制区遗传多态性

目的研究中国青海藏族、汉族mtDNA控制区遗传多态性。方法收集69份青海藏族和青海汉族无关人群外周血样本，对其mtDNA控制区进行序列分析，计算多个多态性指标。结合其他民族mtDNA遗传资料，根据Nei法计算包括青海藏族和汉族群体在内的11个群体之间的Fst和Rst遗传距离．进行聚类分析，绘制系统发生树。结果在青海藏族和汉族群体mtDNA控制区中分别发现56和59个多态性位点。Rst遗传距离显示青海藏族人群与各人群之间遗传距离均较远（P〈0．05）；青海汉族人群与西安汉族、蒙古族、长沙汉族等人群之间距离较近（P〉0．05）。结论我国青海藏族和汉族人群mtDNA具有相对独特的遗传特征，其遗传多态性和个体识别力较高，可用于民族起源、迁徙、法医学个体识别等领域研究。     

Quadruplex real-time PCR for forensic DNA quantitation

Forensic DNA quantitation is an important initial step preceding PCR amplification of the STR loci even though information concerning the quality of the DNA is not revealed. A quadruplex real-time PCR (qPCR) assay was developed to quantify four DNA targets: (1) the human RB1 gene in nuclear DNA, (2) the DAZ gene present on the human Y chromosome, (3) the ATPase8 gene present in human mitochondrial DNA and (4) an artificial internal positive control to reveal possible PCR inhibition. Primers labeled with four different fluorophores are used together with a single quencher using the antiprimer quenching-based qPCR method in one reaction, in which the resultant amplicons are less than 127 bp in size. Sensitivity was shown to be less than ten copies for all four targets in the absence of amplification inhibition. The amplification remained sensitive in the presence of an excess of non-human DNA.