人精子体外氧化损伤对线粒体tRNA~(LeuUUR)基因的影响

目的:探讨活性氧(ROS)对人类精子线粒体tRNALeuUUR基因的氧化损伤。方法:采用Percoll梯度离心法筛选具有正常生理功能的精子,作为正常精子模型,并分为损伤组20例和对照组20例,分别加入次黄嘌呤-黄嘌呤氧化酶体系或不予处理,37℃有氧环境中孵育60min。分别提取精子DNA,以Fpg酶切损伤碱基并采用接头介导PCR(LM-PCR)检测线粒体tRNALeuUUR基因的氧化损伤。采用Rhodamine(Rh123)荧光探针标记精子,通过流式细胞仪检测线粒体膜电位,观察精子的功能。结果:与对照组相比,损伤组精子孵育后线粒体膜电位明显降低[(116.27±11.72)%vs(64.00±4.88)%,P<0.05]。Fpg酶切和LM-PCR显示精子线粒体tRNALeuUUR基因损伤。结论:ROS可能通过对精子线粒体tRNALeuUUR基因氧化损伤而影响精子功能(线粒体膜电位明显降低),从而引起不育。     

PCR和ELISA法在检测HBV-DNA、HCV-RNA和结核分枝杆菌中的作用

目的:探讨PCR技术和ELISA法在检测HBV-DNA、HCV-RNA和结核分枝杆菌中的作用,以及与直接涂片法的对比结果.方法:分别用两法对临床确诊的感染病例和非感染者同时进行检测,并进行对比分析.结果:HBeAg阳性者HBV-DNA阳性,检出率为96.8%,HCV-RNA阳性检出率为19.4%.不同标本TB-DNA平均阳性检出率为39.98%.结论:PCR法灵敏度和特异度分别高于ELISA法和涂片查菌法.     

人肿瘤特异抗原MAGE-3基因疫苗的构建和表达

目的：克隆肿瘤特异性共享抗原mage-3基因，制备基于α－病毒复制酶的高效黑色素瘤特异性基因疫苗。方法：用RT-PCR方法从黑色素瘤细胞系LB373中制备mage-3基因，以含α－病毒复制酶的哺乳细胞高效表达质粒pSMART2a为载体，构建重组DNA疫苗。重组子用载体中的T7和T3启动子序列为测序引物进行自动测序。再将鉴定过的重组质粒用脂质体法转化293细胞，用免疫印迹法和免疫组化法鉴定转化细胞中mage-3蛋白的表达。结果：正确构建了mage-3/pSMART2a重组质粒，并且在转化此质粒的293细胞中检测出了mage-3蛋白的表达。结论：此重组mage-3/pSMART2a质粒可以作为肿瘤特异的DNA疫苗，可进行下一步的肿瘤动物模型的疫苗接种及相关免疫学效应研究。     

Usefulness of PCR Strategies For Early Diagnosis of Chagas'' Disease Reactivation and Treatment Follow-Up in Heart Transplantation

Heart transplantation (HTx) is a useful therapy for end‐stage Chaga? cardiomyopathy; however, Chagas reactivation remains a mayor complication. Parasitological methods offer poor diagnostic sensitivity, and use of more sensitive tools such as the Polymerase chain reaction (PCR) is usually necessary. In the present study, reactivation incidence and PCR usefulness for early reactivation diagnosis, as well as for treatment response evaluation during follow‐up, were analyzed using Strout parasite detection test, in 10 of 222 consecutive HTx patients suffering Chagas cardiomyopathy. PCR strategies targeted to minicircle sequences (kDNA, detection limit 1 parasite/ 10 mL blood) and miniexon genes (SL‐DNA, 200 parasite/10 mL) were performed to compare parasite burdens between samples. No patients received prophylactic antiprotozoal therapy (benznidazole). Five patients (50%) exhibited clinical reactivation within a mean period of 71.6 days; positive Strout results were observed in most cases presenting clinical manifestations. kDNA‐PCR was positive 38–85 days before reactivation, whereas SLDNA‐PCR became positive only 7–21 days later, revealing post‐HTx parasitic load enhancement present prior to clinical reactivation development. Reactivations were successfully treated with benznidazole and generated negative PCR results. Results observed in this study indicate the value of PCR testing for an early diagnosis of Chagas reactivation as well as for monitoring treatment efficacy.     

通用引物PCR-SSCP技术分析16Sr RNA基因特征快速鉴定细菌

目的探讨通用引物聚合酶链反应-单链构象多态性(PCR-SSCP)技术在细菌检测中的应用.方法选取9种常见的细菌,采用通用引物对细菌的16SrRNA基因进行PCR扩增,产物进行单链构象多态性分析,并以标准菌株为对照.结果一对引物扩增产物SSCP图谱各细菌之间难以区别;两对引物扩增产物SSCP图谱条带数目、相对迁移率及条带之间的间距存在明显,可相互区别;3种细菌的标准菌株和临床菌株的SSCP图谱完全一致.结论采用针对16SrRNA的通用引物PCR-SSCP技术以标准菌株为对照可方便快捷地检测细菌.     

In vivo quantification of fluorescent molecular markers in real-time: A review to evaluate the performance of five existing methods

With the advent of molecular-targeted fluorescent markers, there is a renewed interest in fluorescence quantification methods that are based on continuous wave excitation and multi-spectral image acquisition. However, little is known about their in vivo quantification performance. We reviewed the performance of five selected methods by analytically describing these and varying input parameters of irradiance, excitation geometry, collection efficiency, autofluorescence, melanin content, blood volume, blood oxygenation and tissue scattering using optical properties representing those for human skin. We identified one method that corrects for variations in all parameters. This requires image acquisition before and after marker administration, under identical geometry. Hence, it is suited for applications where the site of interest can be relocated (e.g. anaesthetized animals and dermatology). For applications where relocation is not possible, we identified a second method where the uncertainty in the fluorescence signal was ±20%. Hence, use of these methods can substantially aid in vivo fluorescence quantification compared to use of the raw fluorescence signal, as this changed by more than 3 orders of magnitude. Since these methods can be computed in real-time, they are of particular interest for applications where direct feedback is critical, as diagnostic screening or image-guided surgery.     

LightCycler实时监测PCR定量分析血清HBV DNA

目的 检验LightCycler实时监测PCR（real-time detection PCR,RTD-PCR)对血清中HBV DNA定量检测的灵敏性和可重复性，探讨HBV血清标志物与HBV DNA定量的关系。方法 HBV定量按深圳匹基公司乙肝PCR荧光检测试剂盒使用说明，对乙肝标志物已明确的773例血清中HBV DNA定量结果进行统计分析。结果 实时监测PCR对血清中HBV DNA定量检测的灵敏性高，可检测低至1000拷贝／ml血清；可重复性好，批间误差＜20％；各种标志物类型的血清HBV DNA含量分布情况表明，HBV血清标志物中HBeAg与HBV DNA含量有明显的关系，一般HBeAg阳性血清HBV DNA含量较高，但也有相当一部分例外。结论 LightCycler实时监测PCR对血清中HBV DNA定量检测灵敏性高可重复性好；仅根据HBV血清标志物往往不能确定乙肝患者HBV DNA复制水平的高低，HBV DNA定量检测具有十分重要的临床意义。     

HLA-B_(27)与急性前葡萄膜炎相关性的临床研究

目的 :应用SSP PCR(Sequencespecialprime polymerasechainreaction)基因检定技术对急性前葡萄膜炎 (a cuteanterioruveitis ,AAU)患者HLA B2 7基因进行检测 ,并且对HLA B2 7阳性与阴性患者临床特征加以分析。方法 :采用SSP PCR基因检定技术检测 98例AAU患者及 82例正常人样本的HLA B2 7基因。并对HLA B2 7阳性与阴性患者临床特征进行观察。结果 :98例AAU患者样本中有 5 7例样本呈HLA B2 7阳性 ,82例正常人样本有 4例样本呈HLA B2 7阳性 ,阳性率分别为 5 8.2 %和 4 .9%。经 χ2 检验 ,χ2 =4 1.33,P <0 .0 0 5 ,二组间有显著差异。HLA B2 7阳性患者多见于男性 ,单眼多见 ,粉尘状KP ,发病时视力下降明显 ,易于复发 ,且并发症少为其特征 ,激素治疗效果佳。结论 :采用SSP PCR基因检定技术测定HLA B2 7快速、简单、准确性高、客观性强 ,值得推广和应用。HLA B2 7与急性前葡萄膜炎有着高度相关性。HLA B2 7阳性患者与阴性患者在临床特征上有着一定程度的差异。