Automatic Capillary Electrophoresis Research Articles

Association Analysis between Genotype and Phenotype of α,β-Thalassaemia Carriers in Huizhou Area of Guangdong Province

To analyze the prevalence, genotype distribution and hematological characteristics of α,β-thalassaemia carriers in Huizhou area of Guangdong Province. 10 809 carriers of simple β-thalassaemia and 1 757 carriers of α,β-thalassaemia were enrolled as our study cohort. The hematological parameters were detected by automated blood cell counters and automatic capillary electrophoresis. Suspension array technology, gap-polymerase chain reaction (gap-PCR) and PCR-reverse dot blot were used for the genotyping of thalassaemia carriers. The prevalence of α,β-thalassaemia in Huizhou area of Guangdong Province was 1.99%. A total of 62 genotypes were detected, and the most prevalent genotype was --SEA/ αα, βCD41-42/ βN (19.29%), the next was --SEA/ αα, βIVS-II-654/ βN (16.73%). Significant differences in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were found between different genotype groups for simple β-thalassaemia and α,β-thalassaemia. Violin plots showed that carriers with co-inheritance of β-thalassaemia and mild α-thalassaemia expressed the lightest anemia, and carriers with co-inheritance of β-thalassaemia and hemoglobin H (Hb H) disease expressed the most severe anemia. There is a high prevalence of α,β-thalassaemia in Huizhou area of Guangdong Province. Because of the lack of specific hematological makers for diagnosis of α,β-thalassaemia, it is necessary to distinguish it from simple β-thalassaemia by genotyping of α- and β-thalassaemia in order to correctly guide genetic counseling and prenatal disgnosis.

MLVA-TYPING OF CLINICAL STAMPS OF GENETICALLY CHANGED VIBRIO CHOLERAE BIOTYPE EL TOR INSULATED IN RUSSIA AND UKRAINE IN THE PERIOD OF SEVENTH PANDEMIC CHOLERA

Aim. Conduct in a comparative aspect MLVA-typing of genetically altered cholera vibrio biovar El Tor, isolated from patients during the epidemic (1994) and outbreaks (1993, 1998) in Dagestan with isolates in Mariupol (Ukraine) in 1994-2011 in Moscow (2010, 2012), India (1964, 2006, 2007), Bangladesh 1991, 1994, 2001, 2004) and to establish Phylogenetic connections between strains of cholera vibrios isolated in different years in these territories, to ascertain the source of their drift. Materials and methods. MLVA-tyP-ing was carried out in PCR at 5 variable loci of 35 clinical strains of genetically modified Vibrio cholerae byotyPe El Tor. The obtained amPlicon was studied in the system of automatic caPillary electroPhoresis ExPerion («Bio Rad Laboratories», USA). For Phylogenetic analysis, along with MLVA-genotyPes, 35 strains of Vibrio cholerae from the Institute's collection used Published genotyPes of strains isolated in India, Bangladesh, Haiti. Results. The investigated strains of cholera vibrio are referred to 21 MLVA-tyPes, divided into 2 main clades and 1 seParate branch with clonal clusters and subclusters, each of which contains closely related strains of cholera vibrio genovariants having a different degree of Phylogenetic relationshiP - full or Partial identity of allelic Profiles of five variable loci. The sources of drift of genetically modified Vibrio cholerae byotyPe El Tor to Russia and Ukraine from disadvantaged cholera of India, Bangladesh, Azerbaijan and the countries of the Middle East have been established. Conclusion. The obtained data testify to the PolymorPhism of MLVA-tyPes of genetically altered strains of cholera vibrio of the biologist El Tor, evolved in different years and caused ePidemics or outbreaks of cholera in different territories during different time Periods of the course of the seventh cholera Pandemic, and also suggest the Polyclonal origin of the Vibrio cholerae biovar El Tor and the source of their drift to the territory of the Russian Federation and Ukraine.

Value of Analyzing Hemoglobin A2 by ROC Curve for Screening Thalassemia

To investigate the value of hemoglobin A2(HbA2) for screening thalassemia. A total of 2 000 adults' peripheral blood samples from Guangdong Women and Children Hospital from June 2013 to January 2014 were collected. The hemoglobin A2 (HbA2) level was analyzed by the full automatic capillary electrophoresis technique, and the genotypes of thalassemia were detected. The optimal cutoff values of HbA2 for screening silent α-thalassemia, α-thalassemia trait, intermedia α-thalassemia and β-thalassemia trait were 2.85%, 2.65%, 2.25% and 3.45%, respectively; the areas under receiver operator characteristic (ROC) curve were 0.709, 0.839, 0.979 and 0.997 respectively; the sensitivities were 0.481, 0.721, 0.953 and 0.994, and the specificities were 0.846, 0.837, 0.929 and 0.969 respectively. The optimal cutoff values of HbA2 for screening different type of thalassemia based on our laboratory data are established by using ROC curve. According to the area under ROC curve, a satisfactory accuracy for screening intermedia α-thalassemia and β-thalassemia trait can be achieved by detecting hemoglobin A2 level.

Neonatal screening for α‐thalassemia by cord hemoglobin Barts: how effective is it?

It has long been recognized that the hemoglobin (Hb) Bart's in cord blood is an accurate indicator of α-thalassemia and that the level of Hb Bart's was increased accordingly with the increasing numbers of the defective α-genes. This study used an automatic capillary electrophoresis system to determine the Hb Bart's levels in cord blood. Molecular analyses were used to detect various genotypes of α-thalassemia. Sixty-nine of the total 1169 newborns were found to have an increased Hb Bart's in cord blood, in whom the diagnosis of α-thalassemia was confirmed by the DNA analysis. The remaining 1100 newborns had no detectable Hb Bart's at birth; among these, 45 carriers of silent α-thalassemia were diagnosed by DNA analysis. All the 45 cases had only the -α(3.7) deletion genotype. For newborns of one α-gene mutation, especially for 3.7-kb deletion, the method based on Hb Bart's is inadequate and is therefore not reliable for screening.

MLVA를 이용한 경북지역 소에서 분리된 Brucella abortus의 유전형별

Brucella (B.) abortus is a facultative intracellular pathogen that infects a wide variety of animal species and human. Brucellosis is the zoonosis and an extremely important disease around the world. Although the eight species can be differentiated by conventional phenotypic tests, these species display a high degree of DNA homology in DNA-DNA hybridization assay (>90%). Various methods have been established for genotyping of Brucella species, but most of analytical methods are lack reproducibility and limited capability to differentiate them. The attempt of this study was to evaluate multiple-locus VNTR analysis (MLVA) for use of epidemiological trace-back analysis in bovine brucellosis. Ninety-four B. abortus isolates from Gyeongbuk province during 2006~2010 were analyzed using 16 VNTR locus. High resolution automatic capillary electrophoresis system was used for more throughput, simpleer, faster, and better discriminable than conventional gel electrophoresis. As a result, 13 different genotypes were identified from 94 B. abortus isolates. MLVA could contribute to epidemiological trace-back analysis of bovine brucellosis.

Quantitative analysis of Hb Bart’s in cord blood by capillary electrophoresis system

It has long been recognized that the presence of hemoglobin (Hb) Bart's in newborn's blood is associated with α-thalassemia. However, the automated high-performance liquid chromatography or low-performance liquid chromatography system is unable to quantify the amount of Hbs Bart's and H, which are eluted at the retention time close to 0min. This study used automatic capillary electrophoresis (CE) system to diagnose various types of α-thalassemia in 587 cord blood samples, including 429 normal α-globin genotype, 120 cases of thalassemia with one α-globin gene defect, 34 cases with two α-globin genes defect, and four cases with three α-globin genes defect. The result showed that the level of Hb Bart's in cord blood was increased accordingly with the increasing numbers of the defective α-globin genes. In addition, Hb Bart's level at 0.2%, as measured by CE, can be used as a cut-off point for α-thalassemia diagnosis in newborns.

Rapid diagnosis of thalassemias and other hemoglobinopathies by capillary electrophoresis system

Basic diagnosis of hemoglobinopathies can be performed by analysis of erythrocyte indices as well as by the separation and quantification of the common hemoglobin (Hb) fractions Hb A(2), Hb S, Hb C, Hb D, Hb E, and Hb F. This study used an automatic capillary zone electrophoresis system to diagnose various types of hemoglobinopathies common in the Thai population. A total of 459 adults were recruited, which consisted of normal, various types of thalassemia carriers, and thalassemia patients with different genotypes. Hb types and quantification of all Hb components were determined by an automated capillary zone electrophoresis. The automatic capillary electrophoresis system can separate and quantitate Hbs A, F, E, A(2), Constant Spring (CS), H, and Bart's in a way that is comparable with other Hb analysis methods. Moreover, the Hb A(2) peak can be distinguished clearly from the Hb E peak in individuals who carry Hb E. The slightly increased levels of Hb A(2), 3.5% +/- 0.4%, which is shown in the carriers of Hb E, confirm that Hb E is the silent phenotype of beta(+)-thalassemia.

Capillary electrophoresis of oligonucleotides using a replaceable sieving buffer with low viscosity-grade hydroxyethyl cellulose

Short model homo-oligomeric deoxynucleotides ranging in length from 12- to 24-mer were separated using a 20 mM Tris(hydroxymethyl)aminomethane-N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid buffer containing 4% hydroxyethy cellulose of low viscosity as the polymer additive pH 7.0). The separation was performed using a DB-17-coated capillary. The influence of instrumental parameters such as field strength and temperature was evaluated. It required a separation voltage of 12 kV at reversed polarity and a temperature of 25°C. Efficiencies of up to 2.5×106/m were obtained. The most important parameters influencing the separation were the concentration and the viscosity of the polymer used. Different viscosity grades of hydroxyethy cellulose were evaluated for their loading time on the capillary and their ability to separate p(dA)12–18 and p(dA)19–24. The use of low viscosity-grade hydroxyethy cellulose at a relatively high concentration (4%) made it possible to replace the buffer after every run and permitted the use of hydrodynamic injection of oligonucleotide samples. The entangled polymer solution system was found to be applicable on automatic capillary electrophoresis (CE) equipment. For quantitation, the use of an internal standard has been shown to improve both migration time and peak area repeatability. This method using low viscosity-grade hydroxyethy cellulose has been demonstrated to have the repeatability, linearity and selectivity required for stability studies of oligonucleotides.