鉴别外周血与月经血的六重mRNA荧光复合检测体系构建

目的基于外周血、月经血中特异的mRNA分子标记,建立一种能有效鉴别外周血与月经血的六重mRNA荧光复合检测体系。方法通过文献及数据调研,筛选出在外周血和月经血中特异性表达的mRNA标记分子,设计并验证引物。经过总RNA提取、逆转录、多重PCR(MPCR)扩增、遗传分析仪电泳检测分析等步骤,构建一个基于毛细管电泳技术的六重mRNA荧光复合检测体系,并使用38份样本对体系进行检测验证。结果在外周血样本中检测到外周血标记(HBB、HBA)和管家基因(GAPDH、ACTB)的表达;在月经血样本中检测到所有标记(HBB、HBA、MMP7、MMP10、GAPDH、ACTB)的表达;在非血样本中只检测到管家基因(GAPDH、ACTB)的表达。结论本研究构建的六重mRNA荧光复合检测系统可以有效鉴别外周血与月经血样本。     

线粒体16srRNA和ND4基因在种属鉴定中的应用研究

目的构建一种用于种属鉴定的线粒体DNA(m tDNA)16 srRNA和ND4基因荧光标记复合扩增检测体系。方法利用引物设计软件(Prim er 5)对两个m tDNA序列ND4基因和16 srRNA基因设计两对引物,每对引物中的一条在5’端标记荧光素(6-FAM)。按传统复合扩增技术建立复合扩增体系,用AB I PR ISM 310基因分析仪对产物进行分析。结果人类DNA扩增产物出现两个峰,片段大小分别为110bp的人类特异片段和149bp的人与动物共有片段,而动物DNA扩增产物出现一个峰,片段大小为149bp。对30个实验室存放5~15年的陈旧人血痕也能明确判断其种属来源。结论该体系可以明确区分人源性生物检材与其它常见动物样本,对实验室长期存放的陈旧检材也具有较好的检测能力。     

GC/MS衍生化测定精浆中游离支链氨基酸和果糖

目的 建立人精浆中支链氨基酸和果糖的气相质谱衍生化分析方法.方法 精液样本液化离心后,按精子活率的临床标准分为正常组和非正常组,经TMS微波衍生化,GC/MS分析两组支链氨基酸、果糖衍生物含量差异.结果 正常组与非正常组的缬氨酸,亮氨酸,异亮氨酸在相对含量上均有一定差异.正常组3种氨基酸相对含量高于非正常组.果糖分析观察到同样的特征,正常组相对含量高于非正常组.结论 精子活率低的精浆样本仍可检出支链氨基酸和果糖,其差异有助于无精子、少精子精斑的法医学鉴别.     

人类岩藻糖基转移酶5特异性分布及其在精细胞的表达与定位

目的研究人类岩藻糖基转移酶5(fucosyltransferase 5,FUT5)的特异性分布及在精细胞的表达与定位。方法收集健康志愿者的精液(分离精细胞并提取精细胞膜蛋白)、阴道拭子、唾液及静脉血,应用免疫印迹方法检测FUT5在人类精细胞膜、精浆、阴道液、唾液及血清中的表达量,采用免疫荧光技术检测FUT5在精细胞中的表达与定位。结果免疫印迹方法结果显示FUT5在精细胞膜及血清中有较高表达,但在精浆、阴道液及唾液中未被检测到。免疫荧光实验结果显示FUT5主要存在于精细胞头部。表明人类精细胞膜存在一定表达量的特异性FUT5,可采用抗原-抗体反应分离精液和阴道液混合斑中的精细胞。结论人类FUT5表现出分布特异性,可应用到法医学性犯罪案件中混合斑的鉴定。     

荧光标记STR分型技术检验腐败组织基因型

探讨腐败组织荧光标记STR分型检测技术的应用价值。应用含12个STR基因座及一个性别基因座的2个荧光标记的复合扩增系统,对40例1～6周的腐败肌肉提取的DNA进行扩增,用变性聚丙烯酰胺凝胶电泳,PE377测序仪分析基因型。所检测样本在12个STR基因座均扩增出特异性谱带,并可判定其基因型。荧光标记STR检测技术对腐败组织分型可靠,在实际检案中具有较高的应用价值。     

血痕特异性mRNA标记研究

血痕是案发现场尤其是命案中比较常见的生物物证,而血痕的正确组织来源推断是当前鉴定工作中急需解决的问题。随着法医物证学的不断发展,以mRNA(messenger RNA)为基础的体液斑迹组织来源鉴定技术作为一种不同于传统血痕免疫学检测的新型方法,已经越加显示其独特的优越性。在该技术的基础上,实现共同提取生物物证的RNA与DNA的目标,建立体液斑迹鉴定与DNA分型兼容的方法,有利于现场重建,提高生物物证的证据效力,完善证据链。本研究建立了一个包括血痕总RNA提取、逆转录、荧光特异引物扩增、遗传分析仪电泳检测分析等步骤的血痕来源推断技术平台。实验共采集制备了40份的中国人群(女性)外周血、16份月经血样本,筛选了5个外周血标记:HBA、HBB、GYPA、SPTB、ALAS2,2个月经血标记:MMP7、MMP11,构建了一个囊括外周血、月经血特异标记的荧光复合扩增体系。结果显示mRNA技术为基础的鉴定血痕来源的方法是可行的,并且建立了血痕RNA检验的遗传分析仪结果判读方法。     

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Primer 5．0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。     

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。     

Penta E基因座稀有等位基因32的确认

1 案例资料 1.1 简要案情 本鉴定中心日常检案中一起亲子鉴定案件,案件样本为血液样本. 1.2 初次检验 采用chelex-100方法提取血液样本DNA,使用AGCU EX22人类荧光标记STR复合扩增检测试剂(无锡中德美联生物技术有限公司)在9700扩增仪(美国Applied Biosystems公司)上进行复合...     

The detection of inherited enzyme polymorphism in semen (author's transl)]

Our investigation of the occurrence of the enzymes phosphoglucomutase (PGM), glutamate-pyruvate-transaminase (GPT), adenylatkinase (AK), adenosine-desaminase (ADA), and 6-phophogluconate-dehydrogenase (6-PGD) produced the following results: The phosphoglucomutase type was demonstrated in the most sperm samples and seminal stains in accordance with the corresponding blood type. This enzyme is rather stable and could still be demonstrated well in 1-month old stains. The glutamate-pyruvate-transaminase can only seldom be determined in semen and seminal stains. We only found the GPT 1 type, which is known to have usually the strongest activity. The adenylatekinase was demonstrable in the most fresh ejaculates (not older than 24 h) and in about half the seminal stains (not older than 7 days)--The AK--2-band gets weak with increasing lay days, which may lead to incorrect determinations. The adenosine-desaminase could not be determined in sperm. On the contrary, 6-phosphogluconate-dehydrogenase could be demonstrated in fresh semen samples and also partly in seminal stains up to 7 days. The demonstration of the enzymes did not depend in any system on the secretor type.     

用等电聚焦酶免疫分析法检测人类精斑GC表现型

本文用IEF结合使用过氧化物酶标记第二抗体的酶免疫分析法检测了17名键康成年男性的精液(斑)和唾液斑及10名健康成年女性的阴道分泌液中的GC表现型。结果发现17份人类精斑均可测出三种GC蛋白普通表现型。在10份阴道液中测出一份样本的GC表现型,3份样本有不甚清楚的GC带,不能定型,其他样本均无GC带。17份唾液斑未测出GC。本法的灵敏度(0.675ng)比文献报道的用过氧化物酶标记第二抗体的酶免疫分析(5.6ng)高。     

A dot-blot-immunoassay for semen identification using a polyclonal antibody against semenogelin, a powerful seminal marker

Among various seminal plasma proteins, semenogelin (Sg), produced in the seminal vesicle, has been considered a candidate for demonstrating the presence of semen. Sg consists of two proteins, one 52 kDa (Sg-I) in size, and the other a mixture of 71 and 76 kDa proteins (Sg-II). Recombinant Sg-I and Sg-II proteins were obtained using a baculovirus system and then injected into a rabbit to produce the respective antibodies [Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells, Int. J. Mol. Med. 2 (1998) 693]. When liquefied seminal plasma was immunoblotted with the anti-Sg-I and Sg-II antibodies, the anti-Sg-II antibody identified a wider range of the polypeptides originating from Sg than did the anti-Sg-I antibody. A dot-blot-immunoassay using anti-Sg-II antibody revealed a clear immunoreactive spot even when the semen was diluted 6400-fold. However, this assay showed that the Sg antigen was undetectable in saliva, urine, vaginal secretions, sweat, nasal secretions and serum. To determine the stability of Sg antigenic activity, filter paper with dried semen stains were kept at 37, 4 and 22 degrees C for 1, 6 and 18 months, respectively, and the Sg antigenic activity was examined. The activity was detectable in an area not less than 0.5 cm x 0.5 cm under all of the above environmental conditions during each period. Finally, semen was mixed with saliva or blood at various volumetric ratios, and used as a source of dried stains. The Sg antigenic activity was detectable in the stains until the ratio of semen to saliva or blood reached 1:8. These results suggest that Sg may be useful as a marker for semen identification.     

用ELISA检测精液及精斑中精子抗原的研究

本实验应用单克隆抗人精子抗体和酶标记羊抗人精子抗体,采用ELISA方法确定精子抗原成份的存在。对10份新鲜精液,15份精斑进行了验测,其结果阳性率为100%。新鲜精液(精子数约10,000万个/ml)稀释100万倍,精斑浸出液稀释50万倍,均可出现阳性。对唾液斑、尿斑、乳汁斑、阴道斑、汗斑及输精管结扎的精液均为阴性。实验结果表明,本法检验精子抗原具有灵敏度高,特异性好的优点。     

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.