Y chromosome detection by Real Time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma

Objectives To validate the use of Real Time PCR, a widely used technique that can detect very low levels of Y chromosomal sequence, and to assess the use of a highly sensitive PCR technique, pyrophosphorolysis-activated polymerisation (PAP), for fetal sex determination using free fetal DNA (ffDNA). Methods The fetal sex was determined by Real Time PCR in 58 pregnancies using ffDNA isolated from maternal plasma. In parallel with the Real Time PCR experiments, the presence of Y chromosome sequence was also determined using PAP on 54 isolated ffDNA samples. Results Both techniques detected Y chromosome sequence at very low levels with 98% specificity and 100% sensitivity (Real Time n = 44, PAP n = 54). Furthermore, the PAP technique was shown to be more robust than the Real Time PCR as none of the samples tested failed to meet the acceptance criteria. Combining the two techniques for male fetal sex detection from maternal blood plasma increases the sensitivity and specificity to 100% in this series. Conclusions This study shows that both Real Time PCR and PAP can be used for Y chromosome detection on ffDNA. Furthermore, by using PAP in combination with Real Time PCR more reliable early prenatal sexing can be performed using ffDNA. Copyright © 2007 John Wiley & Sons, Ltd.     

苯甲酸盐厌氧驯化体系中三氯乙烯的还原脱氯特性

采用气相色谱法实时监测了厌氧条件下,以苯甲酸盐为唯一碳源的驯化活性污泥体系对三氯乙烯(TCE)的还原脱氯情况;同时,结合454焦磷酸测序和实时荧光定量PCR技术对该体系中的微生物群落结构及脱卤拟球菌(DHC)的数量进行分析.结果表明,该驯化体系在94 d内能将TCE完全去除,最终转化产物为一氯乙烯(VC),同时伴随大量的甲烷产生;体系中微生物的多样性很高,涵盖了16个门、33个纲、52个目、88个科和129个属,而且体系中约有51.2%的微生物的分类地位尚未明确,说明体系中还存在很多未知的功能菌;体系对TCE的降解过程其实就是还原脱氯菌与其它功能菌相互作用的结果,且起主要还原脱氯作用的是携带tce A功能基因的DHC.     

Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued. Copyright © 2003 John Wiley & Sons, Ltd.     

亚硝态氮胁迫下菲律宾蛤仔实时定量P CR内参基因的筛选

为了更好地从基因角度研究亚硝态氮对贝类的生态毒性,需要筛选一个合适的内参基因作为参考,对其毒性相关基因进行定量表达分析。本研究以菲律宾蛤仔(Ruditapes philippinarum)作为研究对象,以亚硝态氮胁迫的鳃组织cDNA作为模板,利用geNorm软件和NormFinder软件对6个候选内参基因进行表达稳定性分析,并对2个结果做了综合赋权分析。结果显示,geNorm分析的基因表达稳定性结果为ActinCyPAEF1αUbi18STubu,NormFinder软件分析的结果为ActinUbiCyPAEF1α18STubu,最后经加权赋值法综合分析获得6个候选基因的表达稳定性为ActinCyPAUbiEF1α18STubu,即Actin为表达最稳定的基因。因此,Actin可以作为衡量亚硝态氮胁迫后菲律宾蛤仔鳃组织中基因表达的内参基因。     

生物强化系统微生物分子诊断技术的应用及新发展

现代分子生物技术的飞速发展及其在环境研究领域的应用，为生物强化技术的研究和发展提供了新方法和新思路。本文从生物强化系统特异微生物检测及定量化技术、生物强化系统微生物群落结构组成及动态演替规律研究、生物强化作用机制的分子生物学解析、生物强化菌的基因工程构建、生物强化系统微生物的安全释放及控制技术几方面，对生物强化系统微生物分子诊断技术的应用和发展作了较为全面的综述。     

进水浓度或HRT变化时微好氧处理微生态机制研究

考察改变进水COD浓度（11 000、 15 000、 30 000 mg/Ｌ）或水力停留时间（HRT为42 h、 25 h）对模拟高浓度有机废水微好氧连续小试处理效果的影响,并采用荧光原位杂交-流式细胞术（FISH-FCM）、聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE) 等分子生物学技术及Biolog　FF微孔板法分析了不同处理阶段稳定期曝气柱污泥的微生态变化,以探讨进水COD浓度或HRT改变时微好氧处理效果变化的微生态机制.结果表明,在全部4个处理阶段,曝气柱污泥中酵母含量均保持在＞99.9％的水平.随着进水COD浓度的上升,污泥浓度由2.0 g/Ｌ上升到7.3 g/Ｌ,COD比去除速度由2.3 kg/（kg·d）下降到1.7 kg/（kg·d）,曝气柱污泥真菌群落结构多样性指数由2.05上升至2.19,代谢多样性指数由4.42上升至4.45;而随着HRT下降,污泥浓度从7.3 g/Ｌ下降至6.0 g/Ｌ,COD比去除速度从1.7 kg/（kg·d）上升至2.8 kg/（kg·d）,真菌群落结构多样性指数和代谢多样性指数则分别从2.19和4.45降至0.79和4.36.作为提高进水有机负荷的主要措施,提高进水COD浓度和缩短HRT对于废水处理效果和曝气柱微生态存在截然相反的影响.