Microfluidic chip-based method for genotyping microsatellites,VNTRs and insertion/deletion polymorphisms

We have developed a method to genotype variable number of tandem repeats (VNTRs) and insertion/deletion polymorphisms using an integrated microfluidic chip-based system. We used this method to analyze a) a highly polymorphic pentanucleotide repeat (CCTTT)(n) locus within the 5'-putative promoter region of the human inducible nitric oxide synthase gene (iNOS5) which is associated with diabetic complications and infectious diseases; b) a bi-allelic 27 bp VNTR region within intron 4 of endothelial nitric oxide gene (eNOS27) which is associated with hypertension in type 2 diabetes patients with coronary heart disease and excess risk of advanced diabetic nephropathy in type 1 diabetes patients and c) an insertion/deletion polymorphism within the gene encoding angiotensin-converting enzyme (ACE/ID) which is associated with cardiovascular pathology and nitric oxide activity, and is in strong linkage disequilibrium with functional variants. Following amplifications, samples were mixed with gel-dye and markers and loaded into commercially available microfluidic chips designed for DNA sizing applications. In the study (N = 230), 95 (41%) of the DNA samples were homozygous and 135 (59%) were heterozygous for the iNOS5 repeats. For eNOS27, 173 (75%) of the genotyped DNA samples were homozygous for the larger 4b allele and the remaining 57 samples (25%) were heterozygous (4b/4a). No DNA samples were homozygous for the shorter 4a allele with four 27 bp repeats. In case of ACE/ID, 47 (20%) of the DNA samples were homozygous for the insertion, 65 (28%) were homozygous for the deletion and the remaining 118 (51%) were heterozygous. The results obtained were verified by analyzing random amplicons using bi-directional sequencing and GeneScan 3.0 analyses with 100% concordance being observed. Using the microfluidic chip-based method, separation and DNA sizing and genotyping are rapidly accomplished. The DNA fragments are resolved clearly and the system allows quantitation. Finally, the microfluidic chip-based method may be used for both large- and small-scale genotyping studies.     

应用序列特异引物PCR法对广西壮族HLA－DQA_1进行基因分型

应用序列特异引物多聚酶链反应（ＰＣＲ）方法对８２名无血缘关系的广西壮族健康人的ＨＬＡ－ＤＱＡ＿１进行基因分型。共检出７种ＤＱＡ＿１等位基因，以ＤＱＡ＿１＊０３０１的频率最高，达３１％，其次为ＤＱＡ＿１＊０１０４，０１０２和０５０１，检出率分别为２４．３％，１６．９％和１１．７％。未检出的等位基因有ＤＱＡ＿１＊０２０１，０３０２和０６０１。结果表明，壮族的ＨＬＡ－ＤＱＡ＿１等位基因频率分布除与中国南方人相似外，尚有其特点。序列特异引物ＰＣＲ方法不需要放射性同位素，简便快速、准确可靠的ＨＬＡ－ＤＱＡ＿１基因分型新方法。     

Molecular epidemiology of tuberculosis: toy or tool? A review of the literature and examples from Central Europe

Genotyping has become an indispensable tool in medical microbiology and epidemiology. One of the first targets has been Mycobacterium tuberculosis. Over the past 15 years approximately 900 pertinent publications have substantiated the value of the genotyping approach for tuberculosis control. New insights into the understanding of the natural history of tuberculosis, especially regarding the frequencies of reactivation, reinfection or multiple infection entailed adaptations of pathophysiological concepts. However, assessment of recent transmission, outbreak analysis, and detection of laboratory contamination still form the genuine scope of genotyping. Detection of unsuspected clusters of cases can provide clues to search for further, undetected cases. Uncovering false positive cultures spares the risks and costs of unnecessary treatment and may reveal systematic laboratory weaknesses. Several European countries already profit from nationwide prospective fingerprinting. After providing genotyping results to public health officials, these were able to document epidemiological links for substantially more tuberculosis patients. On a global scale, strain families and particular strains have been identified, characterised and traced in their spread. The importation of Beijing-genotype multidrug-resistant M. tuberculosis into Central European countries will be described here as an example. The goal for further developments is the ability to compare isolates for epidemiological purposes in a single step that also comprises species determination, drug resistance testing and detection of pathogenicity factors.     

产超广谱β-内酰胺酶肺炎克雷伯菌基因型研究

目的：研究医院感染超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌的基因型。方法：收集分离鉴定菌株，通过K—B法药敏实验初筛以确认产ESBLs菌株，并对ESBLs基因进行初步分型。结果：17株肺炎克雷伯菌产生的ESBLs以CTX-M-3、CTX-M-15、CTX-M-22为主要基因型，其中8株为CTX—M-3型；6株为CTX-M-22型；1株为CTX-M-15型；1株同时产CTX-M-3、CTX-M-22；1株同时产CTX-M-15、CTX-M-22型。结论：我院肺炎克雷伯菌临床分离株中的ESBLs基因型以CTX-M型酶为主。     

两种用于人乳头瘤病毒检测方法的比较分析

目的通过对核酸快速导流杂交基因芯片技术与荧光PCR技术检测人乳头瘤病毒的结果进行比较,为临床人乳头瘤病毒的诊断提供恰当的检测方法。方法采用人乳头瘤病毒分型基因芯片检测系统和荧光PCR技术对78例子宫颈癌患者的宫颈脱落细胞进行人乳头瘤病毒检测。结果基因芯片分型检测总阳性率为91.03%(71/78),且均为高危型。单一感染52例,其中HPV16型阳性率为61.54%,HPV18阳性率为11.54%,其他类型感染阳性率26.92%。荧光PCR法检测总阳性率为93.59%(73/78),其中HPV16阳性检出率45.21%,HPV18阳性检出率9.59%。两种检测方法的符合率为97.4%。结论 HPV导流杂交基因芯片技术和荧光PCR技术两者具有良好的符合性,两者都适用于临床检测,荧光PCR技术更适用与宫颈癌的大范围筛查。     

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。