Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders

We report our experience of 14 preimplantation genetic diagnosis (PGD) cycles in eight couples carrying five different single gene disorders, during the last 18 months. Diagnoses were performed for myotonic dystrophy (DM), cystic fibrosis (CF) [ΔF508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously, and two disorders for which PGD had not been previously attempted, namely neurofibromatosis type 2 (NF2) and Crouzon syndrome. Diagnoses for single gene disorders were carried out on ideally two blastomeres biopsied from Day 3 embryos. A highly polymorphic marker was included in each diagnosis to control against contamination. For the dominant disorders, where possible, linked polymorphisms provided an additional means of determining the genotype of the embryo hence reducing the risk of misdiagnosis due to allele dropout (ADO). Multiplex fluorescent polymerase chain reaction (F-PCR) was used in all cases, followed by fragment analysis and/or single-stranded conformation polymorphism (SSCP) for genotyping. Embryo transfer was performed in 13 cycles resulting in one biochemical pregnancy for CF, three normal deliveries (a twin and a singleton) and one early miscarriage for DM and a singleton for Crouzon syndrome. In each case the untransferred embryos were used to confirm the diagnoses performed on the biopsied cells. The results were concordant in all cases. The inclusion of a polymorphic marker allowed the detection of extraneous DNA contamination in two cells from one case. Knowing the genotype of the contaminating DNA allowed its origin to be traced. All five pregnancies were obtained from embryos in which two blastomeres were biopsied for the diagnosis. Our data demonstrate the successful strategy of using multiplex PCR to simultaneously amplify the mutation site and a polymorphic locus, fluorescent PCR technology to achieve greater sensitivity, and two-cell biopsy to increase the efficiency and success of diagnoses. Copyright © 2002 John Wiley & Sons, Ltd.     

One-year survey of opportunistic premise plumbing pathogens and free-living amoebae in the tap-water of one northern city of China

In this study, qPCR was used to quantify opportunistic premise plumbing pathogens (OPPPs) and free-living amoebae in 11 tap water samples collected over four seasons from a city in northern China. Results demonstrated that the average numbers of gene copies of Legionella spp. and Mycobacterium spp. were significantly higher than those of Aeromonas spp. (p?<?0.05). Legionella spp. and Mycobacterium spp. were 100% (44/44) positively detected while P. aeruginosa and Aeromonas spp. were 79.54% (35/44) and 77.27% (34/44) positively detected. Legionella pneumophila was only detected in 4 samples (4/44), demonstrating its occasional occurrence. No Mycobacterium avium or Naegleria fowleri was detected in any of the samples. The average gene copy numbers of target OPPPs were the highest in summer, suggesting seasonal prevalence of OPPPs. Average gene copy numbers of OPPPs in the taps of low-use-frequency were higher than in taps of high-use-frequency, but the difference was not significant for some OPPPs (p?>?0.05). Moderate negative correlations between the chlorine concentration and the gene copy numbers of OPPPs were observed by Spearman analysis (r_s ranged from ? 0.311 to ? 0.710, p?<?0.05). However, no significant correlations existed between OPPPs and AOC, BDOC, or turbidity. Moderate positive correlations were observed between the target microorganisms, especially for Acanthamoeba spp., through Spearman analysis (p?<?0.05). Based on our studies, it is proposed that disinfectant concentration, season, taps with different-use frequency, OPPP species, and potential microbial correlations should be considered for control of OPPPs in tap water.     

纤维载体的生物膜CANON反应器的启动特性

为研究纤维载体在CANON工艺中的运行特性,同时接种亚硝化污泥及厌氧氨氧化污泥启动CANON反应器.结果表明经过85 d运行,成功启动了CANON反应器,NRR从0.09 kg·(m3·d)-1提升至0.9 kg·(m3·d)-1并能稳定运行,说明纤维载体有利于富集污泥,反应器内能维持较高的生物量.随着微生物的富集生长,生物膜变厚,反应器的能力提升,反应器中DO达到5 mg·L-1.利用微电极测得生物膜由表及里的DO梯度为0.32~0 mg·L-1,说明生物膜变厚,氧对生物膜的穿透力减弱,亚硝化微生物量降低.荧光定量PCR结果表明,启动前后NOB菌数量维持在较低水平,AOB菌的丰度增长不大,ANAMMOX菌细胞增长了一个数量级.     

Prenatal diagnosis of autosomal dominant polycystic kidney disease using flanking DNA markers and the polymerase chain reaction

A prenatal diagnosis was carried out on a 9-week-old fetus at risk for autosomal dominant polycystic kidney disease (ADPKD). Ten members of the family were previously typed using five DNA markers linked to the PKD1 locus on chromosome 16, and one marker linked to the putative PKD2 locus on chromosome 2. The polymerase chain reaction (PCR) was used to amplify the D16S125 locus. Pairwise and multipoint lod scores indicated that the family was most likely segregating a PKD1 mutation. The fetus inherited the disease haplotype from the affected parent. Diagnostic accuracy was greater than 99 per cent, taking into account the possibility of genetic heterogeneity.     

食品企业污水中细菌的基因芯片快速检测技术

食品企业污水中含有各种无机污物和有机污物．其中夹带的致病菌将导致多种疾病的爆发和流行．严重威胁着人类的健康。传统的细菌分离、培养和鉴定技术操作繁琐且耗时长（一般需4～7d）。为了快速．准确地检测食品企业污水中存在的细菌，建立了一种采用基因芯片技术对食品企业污水中常见细菌检测和鉴定的实验方法（需时4h）。实验中设计了8对特异引物并成功分成2组混合引物进行多重PCR反应：BI物Ⅰ为Kp＋HlyA＋InvA＋ipaH，引物Ⅱ为Cadc＋Oprl＋Ent＋23SrRNA。效果良好。实验确定了适宜的PCR反应循环数为35个循环，适宜的杂交温度为48℃。用实验制备的基因芯片对模拟水样检测结果的准确率达100％，说明该基因芯片对目的细菌特征基因的检测结果是可靠的。用该方法对食品企业污水水样进行检测具有高准确率，为今后更大规模的检测研究奠定了基础。     

Prenatal diagnosis of fragile X syndrome: Management of the male fetus with a premutation

Direct detection of the fragile X mutation by DNA analysis has greatly simplified prenatal diagnosis of this disease. However, women carrying a fragile X premutation may pass their expanded trinucleotide repeat to sons without expansion to a full mutation. Such sons are predicted to be intellectually normal. In this situation, the accuracy with which the fetal status can be inferred from analysis of chorionic villus sample (CVS) DNA is unclear. We describe such a case, in which it was felt necessary to proceed to fetal blood sampling despite technically unambiguous DNA results from the CVS. The lack of prospective data means that this dilemma may be expected to recur over the next few years when performing prenatal diagnosis on fragile X premutation carriers.     

Applicability of DNA isolated from syncytiotrophoblast vesicles to gene amplification and molecular analysis

Maternal contamination of fetal DNA represents a major problem when highly sensitive molecular techniques are used in the prenatal diagnosis of genetic diseases. For this reason, we have studied the possibility of using DNA isolated from syncytiotrophoblast vesicles as a target of gene amplification (PCR). Three PCR systems were selected which included a repetitive 149 bp fragment of the Y chromosome, the VNTR locus D1S80, and a portion of the β-globin gene. The results of these experiments indicate that DNA isolated from syncytiotrophoblast vesicles is free of maternal contamination and is suitable for gene amplification and DNA analysis.     

广州城市人工水系统中军团菌的发生和分布

综合运用平板分离法、半巢式PCR和EnviroAmp PCR反向杂交检测试剂盒，对广州市部分人工水系统中条件致病菌军团菌属细菌进行分离和检测．结果表明，虽然阳性率随着采样时间和地点的不同而有所差异，但总体来说，广州市人工水系统中存在较高程度的军团菌的污染，中央空调冷却塔水中军团菌阳性率平均为84％，个别路段甚至达到了100%，自来水池中则达到了35％，，但在后者的水样中没有检测到最常见的致病菌嗜肺军团菌．对各种培养方法的优劣及军团菌的季节性发生和分布进行了初步讨论．图2表2参16     

小麦高分子量谷蛋白亚基基因的PCR及AS-PCR标记

选用高分子量麦谷蛋白亚基(HMW-GS)在Glu-D1位点已知的 26个普通小麦品种,包括 15个 (TriticumaestivumL. )川育系列品种及其 11个亲本,根据其亚基Dx5和Dy10基因的特异序列设计引物.通过对Dx5基因的PCR扩增及对Dy10基因的AS-PCR扩增,结果表明,所设计的引物对Dx5和Dy10都能扩增出特异条带,而携有Dx5基因的材料同时也携有Dy10基因.说明用所设计的引物可快速有效的鉴定出普通小麦中是否带有优质的 5 10亚基.图 4参 15