Preimplantation genetic diagnostic protocols for α- and β-thalassaemias using multiplex fluorescent PCR

Haemoglobinopathies including α- and β-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for β-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for β-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting β-thalassaemia mutations (codon 41-42 and IVSI-110) and one for α-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of β-globin and δ-globin was also found to be an important issue that necessitated careful design of primers for the β-globin gene. Copyright © 2001 John Wiley & Sons, Ltd.     

Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

We have developed a new allele-specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig-Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMN^t) gene. An oligonucleotide was designed to be specific to the SMN^t nucleotidic sequence with exonic mismatch G (for SMN^t)→A (for SMN^c) at its 3′ end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMN^t gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre. Copyright © 2001 John Wiley & Sons, Ltd.     

Alpha-thalassaemia prenatal diagnosis by two PCR-based methods

In Cyprus all couples carrying α⁰-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for α-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)_n repeat polymorphic site located between the ψα₁- and α₂-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two α-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)_n repeat polymorphisms analysis. No misdiagnosed cases for α-thalassaemia have been reported to date. Copyright © 2001 John Wiley & Sons, Ltd.     

水传病原微生物PCR-纳米金显色快速检测方法

水传病原微生物是腹泻等肠胃疾病发病的主要原因,而发展快速、有效的检测方法对于控制和减少该类微生物的传播风险十分重要.因此,本文研究了PCR反应结合不修饰的纳米金颗粒(Gold nano-particles,AuNPs)显色检测方法的最佳盐浓度、探针含量、PCR反应条件等,并从纳米金制备、病原菌种类、检测特异性等方面进行考核,确定了最优检测条件为:10μL PCR扩增产物混合20 pmol探针,95℃反应5 min,55.5℃反应3 min后,40℃杂交10 min;此时,先后加入90μL实验用纳米金和一定浓度的PBS溶液,室温观察颜色变化.结果发现,纳米金溶液由红色变为紫色或近于无色为阳性结果,对应的吸光度值迁移出530 nm,以此可达到快速检测病原微生物的目的.     

内循环半短程亚硝化工艺运行条件与微生物群落研究

在内循环半短程亚硝化工艺中,污泥浓度为4 000 mg·L-1、溶解氧小于0.2 mg·L-1、温度(15~29℃)、水力停留时间4.6 h条件下,不同的回流比对系统中微生物群落有着明显的影响,回流比75%时,微生物的生物量达到最高值,出水中的亚硝态氮和氨氮的浓度比可控制在1.定量PCR和16S rRNA基因的克隆文库结果表明:在低溶解氧浓度下氨氧化菌是主要脱氮菌群,该菌群促进了半短程亚硝化反应的进行,与传统的硝化系统比较,在内循环半短程亚硝化工艺中没有检测到硝化螺旋菌(Nitrospira)和硝化杆菌(Nitrobacter),在内循环半短程亚硝化系统中浮霉菌属(Planctomycetes)的量也高于传统的脱氮系统.氨单加氧酶基因克隆文库结果表明,系统中的氨氧化菌群主要属于亚硝化单胞菌属(Nitrosomonas).因此内循环半短程亚硝化工艺在经济和技术上是可行的.     

生物除磷系统中聚磷菌检测常用技术

作为生物除磷系统中的主要功能菌,对聚磷菌菌群进行定性、定量分析是深入理解生物除磷系统、提高除磷效率的必然趋势。目前常用于检测聚磷菌的方法主要有生物化学法和分子生物学法,文章主要阐述了荧光原位杂交技术、聚合酶链反应技术、变性梯度凝胶电泳技术以及多技术结合使用的特点及应用情况,并在此基础上提出了改进措施以及聚磷菌检测技术的发展方向。     

长期施用有机和无机肥对黑土nirS型反硝化菌种群结构和丰度的影响

利用末端限制性片段长度多态性(T-RFLP)和实时荧光定量PCR(real-time quantitative PCR,Q-PCR)技术,结合反硝化潜势(DEA)和土壤理化性质的测定,探索了长期施用有机和无机肥对公主岭黑土nirS型反硝化细菌的群落结构和丰度的影响.试验设不施肥(CK)、单施有机肥(OM)、单施无机肥(NPK)以及有机肥和无机肥混施(MNPK)等4个处理.结果表明,长期施用有机肥显著增加了土壤的DEA,其中OM、NPK和MNPK处理分别为CK处理的5.92、1.81和6.03倍,而NPK和CK间无差异.有机肥处理增加了黑土nirS型反硝化细菌的丰度,OM、NPK和MNPK处理中nirS基因的拷贝数分别为CK的2.73、1.30和3.98倍;NPK处理对nirS基因的拷贝数影响不显著.T-RFLP图谱显示施用有机肥改变了nirS反硝化细菌的群落结构;相比于非有机肥处理,有机肥处理中增加了一类79 bp的片段类型,显著降低了84 bp的片段类型,并完全抑制了一类99 bp的片段类型,而有机肥处理间和非有机肥处理间的nirS群落结构分别相似.系统发育分析表明:黑土中nirS型反硝化菌主要由α、β和γ-变形菌纲及一些尚未培养的微生物组成,79 bp的片段类型与γ-变形菌纲的假单胞菌科(Pseudomonadaceae)和β-变形菌纲的伯克氏菌目(Burkholderiales)相似,84 bp片段类型与Burkholderiales和红环菌目(Rhodocyclales)相似.相关性分析表明,pH、全磷(TP)、全氮(TN)、总有机碳(TOC)、硝态氮(NO3--N)和铵态氮(NH4+-N)依次与nirS型反硝化细菌的种群丰度(r为0.724～0.922,P<0.05)和DEA(r为0.453～0.938,P<0.01)显著相关,DEA与nirS型反硝化细菌的种群丰度显著线性正相关(r=0.85,P<0.01);冗余度分析表明,除含水量外,TN、TP、pH、TOC、NH4+-N和NO3--N(r为0.440～0.862,P<0.01)依次与nirS型反硝化细菌群落结构的变化显著相关,DEA的变化和nirS型反硝化细菌群落结构的变化亦显著相关(r=0.863,P<0.01).本研究表明相比于无机肥处理,公主岭黑土中nirS型反硝化菌的群落结构与丰度对有机肥处理有更显著的响应,且其群落结构的改变与种群丰度的增加与DEA的提高显著相关.