scholarly journals Evaluation of Four DNA Typing Techniques in Epidemiological Investigations of Bovine Tuberculosis

1998 ◽  
Vol 36 (1) ◽  
pp. 168-178 ◽  
Author(s):  
Debby Cousins ◽  
Suzette Williams ◽  
Ernesto Liébana ◽  
Alicia Aranaz ◽  
Annelies Bunschoten ◽  
...  
Keyword(s):  
Restriction Fragment ◽  
Dna Typing ◽  
Rflp Analysis ◽  
Direct Repeat ◽  
Single Copy ◽  
Epidemiological Evidence ◽  
Different Types ◽  
The Republic ◽  
Different Strains ◽  
Typing Technique

DNA fingerprinting techniques were used to type 273 isolates ofMycobacterium bovis from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS6110, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis (J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS6110, and 81.5% of these carried IS6110 on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS6110 identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS6110 was sufficiently sensitive for the typing of isolates with more than three copies of IS6110, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS6110. Spoligotyping may have advantages for the rapid typing of M. bovis, but it needs to be made more sensitive.

scholarly journals Usefulness of Spoligotyping in Molecular Epidemiology of Mycobacterium bovis-Related Infections in South America

1999 ◽  
Vol 37 (2) ◽  
pp. 296-303 ◽  
Author(s):  
Martín J. Zumárraga ◽  
Carlos Martin ◽  
Sofia Samper ◽  
Alicia Alito ◽  
Omar Latini ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
South America ◽  
Mycobacterium Bovis ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Direct Repeat ◽  
South American ◽  
Discriminative Power ◽  
Different Types ◽  
Restriction Fragment Length

Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, whileAluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovisisolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.

scholarly journals Molecular Fingerprinting of Mycobacterium tuberculosisand Risk Factors for Tuberculosis Transmission in Paris, France, and Surrounding Area

1998 ◽  
Vol 36 (2) ◽  
pp. 486-492 ◽  
Author(s):  
M. C. Gutiérrez ◽  
V. Vincent ◽  
D. Aubert ◽  
J. Bizet ◽  
O. Gaillot ◽  
...  
Keyword(s):  
Risk Factors ◽  
Homeless People ◽  
Rflp Analysis ◽  
Direct Repeat ◽  
Single Copy ◽  
Molecular Fingerprinting ◽  
Factors Associated ◽  
Tuberculosis Transmission ◽  
Surrounding Areas ◽  
Large Clusters

Forty-three percent of the tuberculosis cases reported in France are from the Ile de France region. The incidence of tuberculosis in this region is 33 cases per 100,000 inhabitants, twice the national average. A restriction fragment length polymorphism (RFLP) analysis was performed with clinical isolates of Mycobacterium tuberculosis isolated during 1995 in 10 hospitals in Paris and surrounding areas to detect tuberculosis transmission and define the factors associated with clustering in this population. The molecular markers used were the insertion sequence IS6110 and the direct repeat (DR) sequence. Social, demographic, and clinical data were collected from the patients’ medical files. Ten patients with isolates with a single copy of IS6110 were excluded from further analysis. Twenty-four patients with false-positive cultures due to laboratory contamination (based on RFLP analysis with IS6110 and examination of patient data) were also excluded. The study was then conducted with 272 strains isolated from 272 patients. Further fingerprinting was performed by using the DR element with strains with patterns by RFLP analysis with IS6110that differed by one band only and strains with identical patterns by RFLP analysis with IS6110 and with low numbers of copies of IS6110. The combined use of both markers identified unique patterns for 177 strains and clustered 95 (35.7%) strains in 26 groups, each containing isolates from 2 to 12 patients. The clustering was strongly associated with homelessness and the male sex. It was not associated with age, birth in a foreign country, human immunodeficiency virus positivity, or residence in hostels or prison. Isolates from homeless people were often included in large clusters, and homeless people could be the source of tuberculosis transmission for more than 50% of the clustered patients. These results suggest that homeless people play a key role in the spread of M. tuberculosis in the community and that poor socioeconomic conditions are the main risk factors associated with active tuberculosis transmission.

Localization of the Br Polymorphism on a 144 bp Exon of the GPIa Gene and Its Application in Platelet DNA Typing

1994 ◽  
Vol 71 (05) ◽  
pp. 651-654 ◽  
Author(s):  
Rainer Kalb ◽  
Sentot Santoso ◽  
Katja Unkelbach ◽  
Volker Kiefel ◽  
Christian Mueller-Eckhardt
Keyword(s):  
Genomic Organization ◽  
Fragment Length Polymorphism ◽  
Human Platelet ◽  
Dna Typing ◽  
Rflp Analysis ◽  
Serological Typing ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Alloimmune Thrombocytopenia ◽  
Rflp Typing

SummaryAlloimmunization against the human platelet alloantigen system Br (HPA-5) is the second most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. We have recently shown that a single base polymorphism at position 1648 on platelet mRNA coding for GPIa results in an aminoacid substitution at position 505 on the mature GPIa which is associated with the two serological defined Br phenotypes.Since DNA-typing of platelet alloantigens offers possibilities for useful clinical applications, we designed genomic DNA-based restriction fragment length polymorphism (RFLP) typing for Br alloantigens. To establish this technique we analyzed the genomic organization of GPIa adjacent to the polymorphic base. Using the polymerase chain reaction (PCR) of blood cell DNA we have identified two introns (approximately 1.7 and 1.9 kb) flanking a 144 bp coding sequence of the GPIa gene encompassing the polymorphic base 1648. Based on the in- tron sequence, a PCR primer was constructed to amplify a 274 bp fragment which was used for allele-specific RFLP to determine the Br genotypes. The results of RFLP analysis using Mnll endonuclease obtained from 15 donors (2 Br37*, 2 Br^ and 11 Brb/b) correlate perfectly with serological typing by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay.

CRIMINAL LAW IN PROTECTION OF HEALTH FROM THE ABUSE OF DRUGS IN SERBIA

2018 ◽  
Vol 28 (6) ◽  
pp. 1939-1946
Author(s):  
Miodrag Simović ◽  
Dragan Jovašević ◽  
Marina M. Simović
Keyword(s):  
United Nations ◽  
Criminal Law ◽  
International Standards ◽  
Individual State ◽  
Criminal Sanctions ◽  
Criminal Offenses ◽  
Different Types ◽  
The Republic ◽  
To Come ◽  
Practical Sense

Based on international standards adopted within the framework and under the Organisation of the United Nations, all national legislations recognise several different types and forms of criminal acts regarding misuse of narcotics. It is the matter of various activities of unauthorized production, traffic and other forms of inciting or enabling others to come into possession of narcotics for immediate use, which seriously endangers the health and life.Depending on the needs of each individual state, the distinction is made between the offenses, for the perpetrators are given different types and measures of penalties and other criminal sanctions. A similar situation exists in the Republic of Serbia.The paper analyzes the system of criminal offenses in various types and forms of manifestation in the theoretical and practical sense for whose offenders that are prescribed serious criminal sanctions.

СONTRIBUTION OF POLYMORPHIC VARIATION OF ТP53 AND XRCC1 GENES TO THE SUSCEPTIBILITY TO BREAST CANCER FOR WOMEN OF KYRGYZ AND BELARUSIAN NATIONALITY - A COMPARATIVE STUDY ON MULTIFACTOR DIMENSIONALITY REDUCTION METHODS

2018 ◽  
Vol 64 (1) ◽  
pp. 95-101
Author(s):  
Nazira Aldasheva ◽  
Vyacheslav Kipen ◽  
Zhaynagul Isakova ◽  
Sergey Melnov ◽  
Raisa Smolyakova ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Risk ◽  
Cancer Risk ◽  
Dimensionality Reduction ◽  
Multifactor Dimensionality Reduction ◽  
Rflp Analysis ◽  
Kyrgyz Republic ◽  
Increased Risk ◽  
Polymorphic Variants ◽  
The Republic

Basing on Multifactor Dimensionality Reduction method we showed that polymorphic variants p.Q399R (rs25487, XRCC1) and p.P72R (rs1042522, TP53) correlated with increased risk of breast cancer for women from the Kyrgyz Republic and the Republic of Belarus. Cohort for investigation included patients with clinically verified breast cancer: 117 women from the Kyrgyz Republic (nationality - Kyrgyz) and 169 - of the Republic of Belarus (nationality - Belarusians). Group for comparison included (healthy patients without history of cancer pathology at the time of blood sampling) 102 patients from the Kyrgyz Republic, 185 - from the Republic of Belarus. Respectively genotyping of polymorphic variants p.Q399R (rs25487, XRCC1) and p.P72R (rs1042522, TP53) was done by PCR-RFLP. Analysis of the intergenic interactions conducted with MDR 3.0.2 software. Both ethnic groups showed an increase of breast cancer risk in the presence of alleles for SNPs Gln p.Q399R (XRCC1) in the heterozygous state: for the group “Kyrgyz” - OR=2,78 (95% CI=[1,60-4,82]), p=0,001; for the group “Belarusians” - OR=1,85 (95% СІ=[1Д1-2,82], p=0,004. Carriers with combination of alleles Gln (p.Q399R, XRCC1) and Pro (p.P72R, TP53) showed statistically significance increases of breast cancer risk as for patients from the Kyrgyz Republic (OR=2,89, 95% CI=[1,33-6,31]), so as for patients from the Republic of Belarus (OR=3,01, 95% CI=[0,79-11,56]).

scholarly journals Focal Distribution of Baculovirus IE1 Triggered by Its Binding to the hr DNA Elements

2005 ◽  
Vol 79 (1) ◽  
pp. 39-46 ◽  
Author(s):  
Toshihiro Nagamine ◽  
Yu Kawasaki ◽  
Tetsutaro Iizuka ◽  
Shogo Matsumoto
Keyword(s):  
Fluorescent Protein ◽  
Direct Repeat ◽  
Single Copy ◽  
Targeted Mutagenesis ◽  
Homologous Region ◽  
Focus Formation ◽  
Primary Determinant ◽  
Dna Elements ◽  
Species Specific ◽  
Green Fluorescent

ABSTRACT In BmN cells infected with the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), IE1, a principal transcriptional activator, localizes to sites of viral DNA replication. IE1 initially displays focal distribution in BmNPV-infected cells prior to DNA synthesis, whereas the protein expressed by transfection with the ie1 gene is distributed throughout the nucleoplasm instead of localized to discrete subnuclear structures. To identify the inducer of focus formation for IE1, we conducted transfection experiments with an IE1-GFP construct and found that cotransfection with genomic DNA fragments bearing the homologous region (hr) sequences caused the formation of IE1-green fluorescent protein (GFP) foci. The transfection of insect cells with a single plasmid containing exclusively the hr3 sequence and the IE1-GFP gene was sufficient to form IE1-GFP foci. These results suggest that hr elements are a primary determinant of the focal distribution of IE1. An analysis of a series of hr3 deletion mutants showed that a single copy of the direct repeat could induce the formation of IE1 foci. Targeted mutagenesis within the hr-binding domain of IE1-GFP caused impairment of the hr-dependent IE1 localization, suggesting that binding of IE1 to the hr elements is essential for the onset of IE1 focus formation. The observation of BmNPV IE1 foci in non-BmNPV-susceptible cells suggests that no species-specific factors are required for hr-dependent IE1 focus formation.

scholarly journals Population genetic structure of pedunculate oak (Quercus robur L.) in Belarus according to the analysis of chloroplast DNA

2021 ◽  
pp. 59-70
Author(s):  
Vladimir E. Padutov
Keyword(s):  
Genetic Structure ◽  
Chloroplast Dna ◽  
Population Genetic Structure ◽  
Population Genetic ◽  
Quercus Robur ◽  
Phylogenetic Trees ◽  
Rflp Analysis ◽  
Pedunculate Oak ◽  
Maximum Likelihood Methods ◽  
The Republic

Pedunculate oak (Quercus robur L.) is one of the main forest forming species in the Republic of Belarus. Its population genetic structure was formed under the influence of various migration processes. Six chloroplast DNA loci (µdt1, µdt3, µdt4, µcd4, µcd5 and µkk4) were used for the genogeographic study. The material for the analysis was collected in 100 oak forest stands (2325 samples); 18 allelic variants were identified, which are grouped into 17 different combinations (haplotypes). Five of them are widespread (the proportion of occurrence varies from 7 to 48 %, totalling 85 %). The remaining 12 are rare (the proportion of occurrence varies from 1 to 3 %, totalling 15 %). Phylogenetic trees constructed using the nearest neighbour and maximum likelihood methods show the presence of two groups (branches) of haplotypes. One of it comprises 8 variants including 2 dominant haplotypes and the other comprises 9 variants including 3 dominant haplotypes. PCR-RFLP analysis of chloroplast DNA showed that the pedunculate oak in Belarus originates from the Balkan refugium. Haplotype No. 1 (µdt189, µdt3123, µdt4142, µcd494, µcd574, µkk4109) is found almost everywhere in Belarus with the exception of the southwest and northeast, while haplotype No. 8 (µdt189, µdt3121, µdt4142, µcd494, µcd574, µkk4109) is mainly localised in the southwest and northeast. Haplotypes No. 3 (µdt189, µdt3120, µdt4141, µcd494, µcd575, µkk4109) and No. 7 (µdt189, µdt3122, µdt4142, µcd494, µcd574, µkk4109) predominantly found in the west of the country. Haplotype No. 2 (µdt190, µdt3120, µdt4141, µcd495, µcd574, µkk4109) is typical for the southeast.

scholarly journals Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

2000 ◽  
Vol 38 (12) ◽  
pp. 4337-4342 ◽  
Author(s):  
Amalia Georgopoulou ◽  
Panayotis Markoulatos ◽  
Niki Spyrou ◽  
Nicholas C. Vamvakopoulos
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Restriction Fragment Length

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, andAvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, orNcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

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