Rh缺失型D--个体及其家系成员基因分型与序列分析

目的初步探讨Rh缺失型D--个体发生的分子机制。方法采用序列特异性引物聚合酶链反应(PCR-SSP),对两例Rh缺失型D--个体及其家系成员RHD与RHCE基因多个外显子与内含子进行扩增。根据PCR-SSP结果,对所有与血清学表型不符的异常扩增片段进行克隆测序。结果两例Rh缺失型D--个体PCR-SSP扩增后获得D、e的基因相关片段。经测序发现,RhD--个体1第5外显子第22位核苷酸出现缺失,48与90位发生点突变。RhD--个体2第5外显子第89位发生突变。结论Rh基因外显子的缺失以及单个核苷酸的缺失与突变可能是导致Rh缺失型DD--形成的重要原因之一。     

中缅边境间日疟原虫环子孢子蛋白基因分型

根据间日疟原虫环子孢子蛋白(CSP)基因设计特异分型引物,利用巢式PCR技术(Nested-PCR)对采自中缅边境的间日疟患者血样作分型鉴定。检出的174份血样中,PV-I型热带族占54.6%(95/174)、温带族占35.6%(62/174)、PV-II型占2.9%(5/174)、混和感染占6.9%(12/174)。表明中缅边境的间日疟原虫存在4种基因型,以热带族为优势虫株,缅甸拉咱与云南腾冲间日疟原虫CSP基因型构成无差异(χ2=3.381,P0.05)。     

Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses

The genus Capripoxvirus (CaPV) comprises three members namely, sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affecting sheep, goats and cattle, respectively. CaPV infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. Since there are conflicting opinions regarding the host specificity of CaPVs, particularly for goatpox and sheeppox viruses, the development of rapid genotyping tools will facilitate more accurate disease diagnosis and surveillance for better management of capripox outbreaks.This paper describes a species-specific, real time polymerase chain reaction (PCR), based on unique molecular markers that were found in the G-protein-coupled chemokine receptor (GPCR) gene sequences of CaPVs, that uses dual hybridization probes for their simultaneous detection, quantitation and genotyping.The assay can differentiate between CaPV strains based on differences in the melting point temperature (Tm) obtained after fluorescence melting curve analysis (FMCA). It is highly sensitive and presents low intra- and inter-run variation.This real time PCR assay will make a significant contribution to CaPV diagnosis and to the better understanding of the epidemiology of CaPVs by enabling rapid genotyping and gene-based classification of viral strains and unequivocal identification of isolates.     

应用等位基因特异性PCR检测胆固醇酯转运蛋白基因突变

目的:建立检测胆固醇酯转运蛋白(cholesterol ester transfer protein,CETP)基因6种常见突变的等位基因特异性PCR技术。方法:应用针对CETP基因TaqIB(G→A)、I405V(A→G)、D442G(A→G)、R451Q(G→A)、A373P(G→C)和I14A(G→A)这6种常见的突变位点设计的等位基因特异性PCR技术,对海南汉、黎族人群中CETP基因突变类型进行了检测,同时对经上述等位基因特异性PCR检测的样本进行序列测定。结果:在海南汉、黎族人群中,TaqIB(G→A)突变位点可检测出GG、GA、AA3种基因型,I405V(A→G)突变位点可检测出AA、AG、GG3种基因型,D442G(A→G)突变位点可检测出AA、AG2种基因型,但在海南汉、黎族人群中未检测到R451Q(G→A)、A373P(G→C)和I14A(G→A)3种突变类型,用等位基因特异性PCR鉴定的CETP基因突变的基因分型结果与序列测定结果完全符合。结论:等位基因特异性PCR技术操作简便,重复性和稳定性好,可作为鉴定CETP基因突变类型的可行方法。     

水痘-带状疱疹病毒临床分离株基因型别分析以及与Oka疫苗株的区别

目的 运用分子生物学方法 对从水痘或带状疱疹患者皮肤疱疹液中分离得到的水痘-带状疱疹(VZV)株进行基闪型研究,并区分感染是南野牛株还是由Oka疫苗株引起的.方法 从水痘或带状疱疹患者的皮肤水疱液中分离VZV,然后利用聚合酶链反应和限制性片段长度多态性分析对病毒株的ORF38、54、62和R5可变区基因进行分析.结果 在所分离的19株VZV中,存在PstⅠ+ Bgl Ⅰ+ R5A和Pst Ⅰ+ Bgl Ⅰ+RSB两种基因型,其中Pst Ⅰ+ Bgl Ⅰ+ R5A占52.7%,Pst Ⅰ+ Bgl Ⅰ+R5B占47.3%,没有发现与Oka疫苗株相同的基因型.结论 本研究中所分离的VZV毒株均系野生株,它们的基因型与欧洲、美国、日本的VZV分离株均不相同.利用病毒基因组中ORF38和ORF62区域的单一核苷酸多态性,能够区分疫苗株和野毒株.     

Whole genome sequencing of lamb rotavirus and comparative analysis with other mammalian rotaviruses

Rotavirus (RV) epidemiological surveys with molecular analysis of various strains are required for gastroenteritis control and prevention. The lamb rotavirus strain NT, isolated from a diarrhea lamb in China, is considered as a promising vaccine strain. The whole genome of the lamb-NT strain was determined by sequence analysis. Sequence identity and phylogenetic analysis defined the lamb-NT strain as group A, genotype G10P[15]/NSP4[A]/SG1 strain. Comparative genomic analysis of the lamb-NT strain and 17 reference strains reveals that gene reassortments between rotaviruses circulating in different species occurred. Alignment of protein sequences of the genes shows some variations in the important functional regions of VP3 and VP4. These variations are related to host range restriction, virulence, and other potential characters of rotaviruses. Besides, this study also makes a significant foundation for the study of genetic classification, epidemiology, and antigenic diversity of rotaviruses on the molecular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

APOE genotyping: comparison of three methods

Apolipoprotein E (apo E) polymorphism is associated with increased risk of cardiovascular and Alzheimer diseases, making its genotyping of potentially predictive value. We developed a rapid, reliable and specific method for determining APOE genotypes by fluorescent resonance energy transfer (FRET) over a high number of samples in a single run using a LightTyper device and dedicated probes. The method, validated with 75 blood samples, was designed to simultaneously detect three common APOE polymorphisms, epsilon(2,) epsilon(3) and epsilon(4), and to identify in a single reaction any of the six following genotypes: epsilon(2)/epsilon(2), epsilon(3)/epsilon(3), epsilon(4)/epsilon(4), epsilon(3)/epsilon(4), epsilon(4)/epsilon(2), epsilon(3)/epsilon(2). The assay involved three phases: (1) DNA extraction, (2) amplification, and (3) melting curve analysis using FRET technique. Briefly, genomic DNA of patients was extracted from total blood. Fragment of APOE was amplified by a first PCR run. Fluorescent labeled probes were added in a second PCR run. FRET genotyping showed following distribution: (1) 1.3% for epsilon(2)/epsilon(2) and epsilon(4)/epsilon(4) homozygotes, (2) 4.0, 6.6 and 14.7% for epsilon(2)/epsilon(4), epsilon(2)/epsilon(3) and epsilon(3)/epsilon(4) heterozygotes, respectively, and (3) 72.0% for epsilon(3)/epsilon(3) homozygotes. Moreover, a careful analysis of the FRET melting curves allowed us to determine the presence of a new polymorphism on the third position of the codon 158 (-AAGCGT-), namely, two nucleotides downstream from the known polymorphism. When the FRET analysis was compared to those obtained by RFLP and sequencing, the presence of this new polymorphism was confirmed only by sequencing thus indicating that RFLP analysis is not always reliable for genotyping.     

Evaluation of the Nanosphere Verigene System and the VerigeneF5/F2/MTHFR Nucleic Acid Tests

Background

Our ability to detect single nucleotide polymorphisms (SNPs) and gene mutations has become commonplace in the clinical laboratory setting. Molecular genetic testing for gene variants associated with hypercoagulability has become a standard of practice for Factor V and Factor II polymorphisms.

Methods

In this study, we evaluated a novel technology that allows for the routine assessment of these SNPs, the Verigene^® System (Nanosphere Inc, Northbrook, IL), as a low-density array that does not require PCR amplification prior to detection. Precision was assessed by using multiple operators for within and between run performance evaluations. Accuracy was assessed by evaluating 176 DNA samples from patients who had been previously tested for the SNPs of interest in this multicenter study.

Results

No mis-calls were made during the precision studies. Testing of the 176 DNA samples resulted in individual call rates for the F5, F2 and MTHFR genotypes of 98.3%, 94.9%, and 92.6%, respectively.

Conclusions

The Verigene^®F5/F2/MTHFR Nucleic Acid Tests for the Factor V (1691G>A), Factor II (20210G>A) and MTHFR (677C>T) genes were robust methods for SNP detection without the need for DNA amplification. The ease of use and performance of this system makes it suitable for the clinical laboratory setting.     

神经外科重症监护室白色念珠菌基因分型和耐药性研究

目的 分析神经外科重症监护室白色念珠菌的药物敏感情况并建立随机扩增多态性DNA(RAPD)基因分型方法 监测医院感染. 方法 收集自2007年5月至11月从珠江医院神经外科重症监护室内分离的30株白色念珠菌,以微量液基稀释法分析常用抗真菌药物对白色念珠菌药物敏感情况,同时提取DNA后进行RAPD基因分型. 结果 临床分离的30株白色念珠菌对抗真菌药物敏感性分别为伏立康唑(100%)、两性霉素B(100%)、氟康唑(96.3%)、氟胞嘧啶(93.0%)、伊曲康唑(90.0%).30株白色念珠菌RAPD基冈分型共分为27型,没有出现相同的带型. 结论 在白色念珠菌感染的药物治疗中,伏立康唑、两性霉素B敏感性最好,氟康唑、氟胞嘧啶、伊曲康唑表现为较高的敏感性.我院神经外科重症监护室自2007年5月～11月期间并未出现白色念珠菌感染局部暴发流行.