6+1STR试剂盒和电泳凝胶EX-Q20用于DNA快速检验

目的 建立快速个体识别STR分型方法.方法 取采集于FTA上的血样200份,经打孔仪取等量血痕分别使用6+1 STR试剂盒结合新型毛细管电泳凝胶EX-Q20进行快速电泳分析和使用SinofilerTM试剂盒结合POP4胶进行电泳分析,比较两者所耗费时间和结果的差异. 结果 6+1 STR试剂盒结合新型毛细管电泳凝胶能够...     

Determination of CDT,a marker of chronic alcohol abuse,for driving license issuing: immunoassay versus capillary electrophoresis

The present work is aimed at a validation of the carbohydrate-deficient transferrin (CDT) determination in real cases by comparison between the a commercial immunometric method and a method based on capillary electrophoresis. Overall, 650 serum samples from subjects applying to re-obtain their driving license, previously withdrawn for "drunk driving", were investigated. A highly significant correlation (P < 0.001) was found between the results from immunoassay and capillary electrophoresis. However, particularly in the samples with CDT values around the cut-off or moderately elevated, a wide dispersion of the correlation data was found. This finding stresses the need to confirm by alternative techniques all the results from CDT immunoassays. For this purpose, capillary electrophoresis, because of its inherent characteristics of high selectivity, easy operation, high productivity and low operative costs looks well-suited for becoming the method of choice.     

Determination of phosphoglucomutase phenotypes in hair bulbs

The research was to determine a simple method of phosphoglucomutase phenotype identification in hair bulb. The agarose technique and electrophoresis on cellulose-acetate foil methods were chosen because a small quantity of the maternal available for examination. It was found out that 1 or 2 bulbs are sufficient to identify the PGM1 features if the electrophoresis method is applied and if more bulbs are available, the PGM3 characteristic can also be identified. The modified technique was used for staining the phosphoglucomutase phenotypes.     

Increasing Allele Detection by Altering the Quantity of Internal Lane Standard

Electrokinetic injection (EI) is the primary method used in forensic laboratories to load amplified PCR product in capillary electrophoresis for short tandem repeat (STR) fragment separation. Because all samples subjected to capillary electrophoresis use internal lane standard (ILS), this study investigated the consequence of varying the volume of ILS and its effects on allele peak heights and number of alleles detected. Results demonstrated that when the volume of ILS is reduced, the average peak height and number of alleles increased, thereby increasing the sensitivity of the detection method. Sizing anomalies were observed; however, they did not adversely affect accuracy and precision. The method developed in this study offers a simple and universal procedure to increase the alleles detected in forensic STR analysis. Reducing the volume of ILS to achieve greater sensitivity is applicable to all STR amplification kits and capillary electrophoresis instruments currently used in forensic DNA analysis.     

微型DNA指纹图的研究与应用

作者采用小板琼脂糖凝胶电泳,应用MYO探针,Southern印迹杂交技术制作微型DNA指纹图,能获得清晰图谱。实验结果表明,同一个体的血液与血斑,精液与精斑及不同部位的组织,其指纹图谱完全相同。不同个体的微型DNA指纹图谱有明显个体差异。用本法能使大板DNA指纹图的常规检出量重复15～20次,最小检出量为250ng,达到极微量的水平。     

STR基因座等位基因阶梯制备方法尝试

提高PCR-STR分型技术的准确性。采用荧光标记dNTP掺入法,通过DNA自动测序仪分析不同等位基因片段的长度并确定其命名后,建立并比较扩增产物混合-纯化-重扩增法、琼脂糖凝胶电泳-切割回收纯化DNA-重扩增法、非变性聚丙烯酰胺凝胶电泳-切割回收纯化DNA-重扩增法等3种Ladder制备方法。扩增产物混合-纯化-重扩增法相对较简便、快速、经济。Ladder对照使基因分型数据化,判型准确,重复性好,有利于标准化的实现。     

3种甲醛固定组织DNA提取方法的比较

目的对不同方法提取甲醛固定组织中DNA的效果进行比较,寻找一种操作简便、经济实用、质量较高的DNA提取方法。方法取甲醛固定的心肌组织14份,分别以改良酚-氯仿法,改良Trizol法,试剂盒法提取DNA,进行紫外分光光度计测定OD260/OD280值后,经PCR扩增,琼脂糖凝胶电泳分析确定提取的DNA质量。结果改良酚-氯仿法,改良Trizol法,试剂盒法OD260/OD280比值分别为1.841 5±0.380 4、1.370 5±0.336 7、0.831 6±0.175 0。两两比较均有显著性差异(P<0.05)。3种不同方法提取DNA含量分别为0.943 8±0.530 1、0.707 5±0.423 6、0.342 8±0.182 5。PCR扩增后琼脂糖凝胶电泳显示以改良酚-氯仿法所提DNA的谱带清晰度好于其它两种方法。结论改良酚-氯仿法简便有效,所用试剂价格低廉,是一种经济实用的甲醛固定组织DNA提取方法。     

Trizol法大鼠心肌总RNA提取方法探讨

目的 探讨大鼠心肌总RNA的提取方法,为今后法医学研究和实践工作提供参考.方法 用Trizol法,严格注意无RNA酶操作,通过裂解组织,分离、沉淀RNA而提取大鼠心肌总RNA,并用分光光度计、凝胶电泳及RT-PCR检测结果.结果 Trizol法能获得较高纯度和产量的完整总RNA.结论 Trizol法是提取总RNA可靠有效的方法,可望在法医学领域有较广的应用.     

Immunologic characterization of human seminal leucine aminopeptidase (LAP) and its medicolegal use

A new method for identification of seminal stains is described, based on the immunologic demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen and specific for the prostate as well as semen. An antiserum against human seminal plasma was obtained by repeated immunization of rabbits with seminal plasma and Freund's adjuvant. Ouchterlony's double immunodiffusion test and Culliford's precipitin electrophoresis were performed to demonstrate specific proteins of seminal plasma. LAP activity was visualized with L-leucyl-beta-naphthylamide as substrate and with Fast Garnet GBC as coupler. The immunologic analysis of LAP produced two precipitin lines with enzyme activity. One was observed in kidney, jejunum, pancreas, prostate, as well as in semen, and was completely absorbed with kidney homogenates. The other was found only in semen and the prostate and was not absorbed with kidney homogenates. When the anti-seminal plasma serum absorbed with the kidney was used, the semen-specific LAP could be demonstrated by precipitin electrophoresis only in seminal stains stored for up to 2 months, whereas it was not demonstrated in stains from other human body fluids. By means of precipitin electrophoresis the detection of the semen-specific LAP was possible at semen dilutions of up to 1:32. The method described here greatly enhances the value of semen identification and is quite recommendable for the examination of stains in medico-legal practice.     

应用磁珠法提取陈旧骨骼DNA

目的探讨采用磁珠法提取陈旧骨骼DNA的可行性。方法取经土埋或室外暴露下存放1~5年不等的10根长骨,经水洗、刮净,钻取骨密质骨粉3g,应用EQ1000磁珠试剂盒提取DNA,经复合扩增,ABI 3130XL基因分析仪电泳分离,进行STR分型检测。结果 10根长骨均获得完整的STR分型,电泳图谱基线干净,除个别大片段基因座外,等位基因荧光信号分布均衡性较好。结论采用磁珠法提取陈旧骨骼的DNA,能满足分型要求,可在实际检案中选用。     

Detection of A and B antigens of the ABO system in a single-hair stem

A method was elaborated for detection of the system ABO antigens in a single-hair stem, containing no hair bulb or vaginal tunics, through using concurrently electrophoresis in the agarous or agar gel as well as through extracting antigens from a certain gel area onto the gauze with performing subsequently the absorption-elution reaction.     

An efficient method to eliminate streaking in the electrophoretic analysis of haptoglobin in bloodstains

A bloodstain extraction procedure that improves the analysis of haptoglobin in dried bloodstains has been developed. The streaking of electrophoresis gels caused by deteriorated hemoglobin can be eliminated by incorporating chloroform in the bloodstain extraction procedure. The method is easier to execute than previously published techniques for eliminating the adverse effects of deteriorated hemoglobin on the analysis of haptoglobin. Bloodstains up to two years old were correctly phenotyped in haptoglobin by this method.     

Determination of carbohydrate deficient transferrin (CDT) with capillary electrophoresis: an inter laboratory comparison

Carbohydrate deficient transferrin (CDT) is currently the most specific laboratory marker of chronic or sustained alcohol abuse. CDT is increasingly being used as a diagnostic tool in the areas employment, traffic safety and forensic medicine. In recent times, capillary electrophoresis (CE) has been proposed as a convenient tool for rapid, precise and accurate CDT determination, not only for research but also for routine analyses. Quite recently, commercial kits have been introduced which, reportedly, could simplify and standardize CDT analysis with capillary electrophoresis. The present work was aimed at testing the ruggedness of a capillary electrophoretic method based on a commercial kit (CEofix, Analis), by comparing the results obtained with different instruments in different laboratories, on a panel of sera randomly collected and exchanged. The results showed, notwithstanding few outliers, excellent correlation of the results obtained in the two laboratories (R=0.974). Also high concordance was found when results were classified as positive or negative on the basis of a cut-off (1.25%) established from a control group of teetotalers. In conclusion the present data support the usefulness of capillary electrophoresis for CDT determination for clinical, forensic and administrative diagnosis of chronic alcohol abuse.     

Agarose gel electrophoresis of human erythrocyte glutamic-pyruvic transaminase (GPT EC 2.6.1.2)

The method of choice to determine erythrocyte glutamate-pyruvic transaminase (GPT) including rare variants was starch gel electrophoresis. Methods using agarose as gel medium were not reported to our knowledge. We present an adapted method using the Tris/maleate buffer system and an agarose of low endosmosis. The common as well as rare variant types of GPT were quickly and reliably separated. In addition, a method for the consecutive determination of esterase D (ESD) and GPT on the same gel using the malic acid buffer system is described.     

A study of sex identification of trace, dried bloodstains using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe

A new method is discussed which examines trace, dried bloodstains by gel in situ hybridization using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe to determine the sex of the bloodstain for forensic medicine application. The complete DNA is transferred directly by electrophoresis onto the gel intact, bypassing the possibilities of impurities contaminating the sample and of DNA degradation. The method has proven accurate for small (2.5-mm-diameter) samples aged up to eight years and is quick, simple, and easily read.     

The simultaneous electrophoretic analysis of esterase D and phosphoglucomutase subtyping in fresh blood and in dried bloodstains

Phosphoglucomutase1 (PGM) subtyping and esterase D phenotyping were simultaneously performed by electrophoresis of bloodstained fibers using agarose and a Tris-maleic acid buffer system , pH 5.4. This method reduces anodal gel shrinkage and shortens development time when compared to the conventional electrophoretic technique for PGM subtyping which is performed at pH 7.4 using an agarose-starch substrate.     

应用mtDNA16S rRNA基因测序鉴定肉产品种属

目的 应用mtDNA 16S rRNA基因测序方法进行肉产品种属鉴定.方法 15份有关部门送检的未知动物肌肉样本,3份市购猪、牛和羊的肌肉为对照样本.常规酚-氯仿法提取模板DNA,分别用通用引物和特异性引物扩增mtDNA的16S rRNA基因片段,产物用1.5％琼脂糖电泳检测,扩增的基因片段由上海生物工程公司进行序列测...     

Simultaneous typing of C3 and Tf and of Gc and Bf on Cellogel electrophoresis

A method is described to type C3 and Tf on the same Cellogel strip (and Bf simultaneously on a parallel strip). A different method allows to type Gc and Bf on one Cellogel strip. These methods are rapid, easy and reliable, although agarose electrophoresis is a little more effective.     

单细胞凝胶电泳检测大鼠死后肝细胞核DNA降解

目的应用单细胞凝胶电泳技术(SCGE)检测大鼠死后肝细胞核DNA降解规律,分析与死亡时间的关系,为早期死亡时间的推断提供新的方法。方法在大鼠死后30h内,每隔3h取肝组织样本进行单细胞凝胶电泳,用共聚焦显微镜摄取彗星图像,应用彗星图像分析软件(IM I1.0)进行图像分析,并作统计学分析。结果死后大鼠的肝细胞在电泳图像上出现明显的彗星形拖尾,其尾长(TL)、尾矩(TM)在一定的时间范围内(0~18h)随死亡时间的延长而逐渐增大,二者均与死亡时间(PM I)呈现一定的相关回归关系。结论单细胞凝胶电泳技术可应用于早期死亡时间的推断。     

A new method for typing haptoglobin in bloodstains using immobilized allo A lectin

Allo A lectin from the beetle, which is beta-D-galactose specific, reacts to haptoglobin but not to hemoglobin. The use of allo A-Sepharose for typing haptoglobin in bloodstains helped eliminate hemoglobin from the bloodstain extract and presented highly resolved haptoglobin patterns by disc gel electrophoresis. This method is simple and rapid for typing haptoglobin in bloodstains and can be easily used in forensic science laboratories.