Genetic analysis of the hypervariable region of the human astrovirus nsp1a coding region: Design of a new RFLP typing method

2007 ◽  
Vol 80 (2) ◽  
pp. 306-315 ◽  
Author(s):  
Susana Guix ◽  
Santiago Caballero ◽  
Cristina Fuentes ◽  
Albert Bosch ◽  
Rosa M. Pintó
Keyword(s):  
Genetic Analysis ◽  
Hypervariable Region ◽  
Coding Region ◽  
Typing Method ◽  
Human Astrovirus ◽  
Rflp Typing

Rapid Molecular Typing of Methicillin-ResistantStaphylococcus aureusby PCR-RFLP

2001 ◽  
Vol 22 (5) ◽  
pp. 294-298 ◽  
Author(s):  
Thomas A. Wichelhaus ◽  
Klaus-Peter Hunfeld ◽  
Boris Böddinghaus ◽  
Peter Kraiczy ◽  
Volker Schàfer ◽  
...  
Keyword(s):  
Fragment Length Polymorphism ◽  
Restriction Analysis ◽  
Protein A ◽  
Hypervariable Region ◽  
Epidemiological Surveillance ◽  
Typing Method ◽  
Rapid Pcr ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Rflp Typing

AbstractObjective:To establish a new, rapid, and reliable genotypic fingerprinting technique for methicillin-resistantStaphylococcus aureus(MRSA) typing in routine epidemiological surveillance.Design:The method is based on polymerase chain reaction (PCR) restriction fragment-length polymorphism (RFLP) followingHaeII digestion of simultaneously amplified parts of the protein A gene, the coagulase gene, and the hypervariable region adjacent tomecA. A total of 46 MRSA initial isolates were analyzed, including 14 isolates from five countries; the six German epidemic strains; 16 isolates from the Frankfurt metropolitan area, which were known to be heterogeneous by pulsed-field gel electrophoresis (PFGE); and 10 isolates obtained during three epidemics, all of which displayed an identical genotype.Results:Restriction analysis by PCR-RFLP permitted discrimination of 10 of 14 international isolates, all six German epidemic strains, and 15 of 16 national isolates. It also confirmed the homogeneous character of the 10 outbreak isolates.Conclusions:This new and rapid PCR-RFLP typing method is an attractive tool in routine epidemiological surveillance. Its impressive characteristics are ease of performance and interpretation, while at the same time guaranteeing good discriminatory power, reproducibility, and typeability.

F8 genetic analysis strategies when standard approaches fail

2014 ◽  
Vol 34 (02) ◽  
pp. 167-173 ◽  
Author(s):  
B. Pezeshkpoor ◽  
A. Pavlova ◽  
J. Oldenburg ◽  
O. El-Maarri
Keyword(s):  
Genetic Analysis ◽  
Half Life ◽  
Untranslated Region ◽  
Mutation Screening ◽  
Screening Methods ◽  
Haemophilia A ◽  
Splice Sites ◽  
Coding Region ◽  
Coding Regions ◽  
Analysis Strategies

SummaryHaemophilia A is a common X-linked recessive disorder caused by mutations in F8 leading to deficiency or dysfunction of coagulant factor VIII (FVIII). Despite tremendous improvements in mutation screening methods, in a small group of patients with FVIII deficiency suffering from haemophilia A, no DNA change can be found. In these patients, analysis reveals no causal mutations even after sequencing the whole coding region of F8 including the flanking splice sites, as well as the promoter and the 3’ untranslated region (UTR). After excluding the mutations mimicking the haemophilia A phenotype in interacting partners of the FVIII protein affecting the half life and transport of the protein, mutations or rearrangements in non-coding regions of F8 have to be considered responsible for the haemophilia A phenotype.In this review, we present the experiences with molecular diagnosis of such cases and approaches to be applied for mutation negative patients.

A PCR-RFLP typing method for adhesion molecule gene polymorphisms and allele frequencies in a normal UK population

2002 ◽  
Vol 29 (2) ◽  
pp. 109-111 ◽  
Author(s):  
R. A. Gbadegesin ◽  
C. J. Watson ◽  
S. A. Cotton ◽  
P. E. C. Brenchley ◽  
N. J. A. Webb
Keyword(s):  
Adhesion Molecule ◽  
Gene Polymorphisms ◽  
Allele Frequencies ◽  
Typing Method ◽  
Pcr Rflp ◽  
Adhesion Molecule Gene ◽  
Rflp Typing

New mutations in SERPING1 gene of Brazilian patients with hereditary angioedema

2016 ◽  
Vol 397 (4) ◽  
pp. 337-344 ◽  
Author(s):  
Nathália Cagini ◽  
C.L. Veronez ◽  
R.N. Constantino-Silva ◽  
Márcia Buzolin ◽  
Renan Paulo Martin ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Genetic Analysis ◽  
Hereditary Angioedema ◽  
Autosomal Dominant ◽  
Missense Mutations ◽  
Inherited Disease ◽  
Coding Region ◽  
Serping1 Gene ◽  
Brazilian Cohort ◽  
New Mutations

Abstract Hereditary Angioedema is an autosomal dominant inherited disease leading to oedema attacks with variable severity and localization predominantly caused by C1-INH deficit. More than 400 mutations have been already identified, however no genetic analysis of a Brazilian cohort of HAE patients with C1-INH deficiency has been published. Our aim was to perform genetic analysis of C1-INH gene (SERPING1) in Brazilian HAE patients. We screened the whole SERPING1 coding region from 30 subjects out of 16 unrelated families with confirmed diagnosis of HAE due to C1-INH deficiency. Clinical diagnosis was based on symptoms and quantitative and/or functional analysis of C1-INH. We identified fifteen different mutations among which eight were not previously described according to databases. We found five small deletions (c.97_115del19; c.553delG; c.776_782del7; c.1075_1089del15 and c.1353_1354delGA), producing frameshifts leading to premature stop codons; seven missense mutations (c.498C>A; c.550G>C; c.752T>C; c.889G>A; c.1376C>A; c.1396C>T; c.1431C>A); one nonsense mutation (c.1480C>T), and two intronic alterations (c.51+1G>T; c.51+2T>C). Despite the small number of participants in this study, our results show mutations not previously identified in SERPING1 gene. This study represents the first Brazilian HAE cohort evaluated for mutations and it introduces the possibility to perform genetic analysis in case of need for differential diagnosis.

scholarly journals Prevalence of Group A Streptococci in Moroccan Children with Pharyngitis and Emm Type Distribution

2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Sara Himri ◽  
Bouchra Oumokhtar ◽  
Samira Atmani ◽  
Btissam Arhoune ◽  
Kaoutar Moutaouakkil ◽  
...  
Keyword(s):  
Care Center ◽  
Hypervariable Region ◽  
Diffusion Method ◽  
Group A Streptococci ◽  
Sensitivity Testing ◽  
Typing Method ◽  
Disk Diffusion Method ◽  
Health Care Center ◽  
Group A ◽  
Emm Type

Background: Streptococcus pyogenes is responsible for a wide variety of diseases, including noninvasive and severe invasive infections. The emm gene encodes the M protein that is the avirulence factor and immunological determinant of group A streptococci. Emm typing is the group A Streptococci (GAS) standard molecular typing method based on the amplification of the N terminal hypervariable region of the emm gene. Objectives: The aim of the present study was to determine the prevalence of GAS in children with pharyngitis and determine different types of emm gene in the GAS isolates using emm typing. Methods: The study was carried out over a period of 14 months (from February 2017 to March 2018). Throat samples were collected from cases aged ≤ 18 years with pharyngitis referring to a primary health care center in Fez, Morocco. GAS isolates were subjected to conventional tests to confirm species identification. Antimicrobial susceptibility testing was performed using the standard disk diffusion method. We researched emm gene by a polymerase chain reaction (PCR). Emm types were determined by a sequence-based protocol. Demographic and clinical data were recorded from each patient. Results: From a total of 177 throat samples, 11 isolates (6.2%) were identified as GAS in children with pharyngitis. Antibiotic sensitivity testing revealed that all the GAS isolates were sensitive to penicillin. The sequencing of the PCR products of the emm gene revealed that emm90 was the most obtained emm type (30,77%); while emm75 was the least type observed (7.7%). Conclusions: The emm90 is the most prevalent type detected from patients with tonsillitis. Penicillin and erythromycin are still the foremost effective antibiotics to treat GAS pharyngitis.

scholarly journals Characterization of Enterovirus Isolates from Patients with Heart Muscle Disease in a Selenium-Deficient Area of China

2000 ◽  
Vol 38 (10) ◽  
pp. 3538-3543 ◽  
Author(s):  
Tianqing Peng ◽  
Yanwen Li ◽  
Yingzhen Yang ◽  
Cunlong Niu ◽  
Peter Morgan-Capner ◽  
...  
Keyword(s):  
Rural Areas ◽  
Sequence Data ◽  
Laboratory Strain ◽  
Muscle Disease ◽  
Keshan Disease ◽  
Nucleotide Sequencing ◽  
Coding Region ◽  
Typing Method ◽  
Suckling Mice ◽  
Nontranslated Region

An association of enterovirus infection with endemic cardiomyopathy (Keshan disease [KD]) and outbreaks of myocarditis in selenium-deficient rural areas of southwestern China has been established. Enteroviruses have been isolated from patients with KD or during outbreaks of myocarditis in last two decades. Six of these isolates grew readily in cell lines (Vero or HEp-2) and were investigated by a novel molecular typing method apart from serotyping and pathogenicity. A neutralization assay identified two isolates from KD as coxsackievirus serotype B2 (CVB2) and two isolates from myocarditis as coxsackievirus serotype B6 (CVB6) but failed to type the remaining two isolates, also from myocarditis. Direct nucleotide sequencing of reverse transcription-PCR products amplified from the 5′ nontranslated region (5′NTR) of these viruses confirmed that they belong to a phylogenetic cluster consisting of coxsackie B-like viruses, including some echovirus serotypes. Sequence analysis of the coding region for viral capsid protein VP1 showed that two isolates serotyped as CVB2 have the highest amino acid sequence homology with CVB2 and that the remaining four isolates, two CVB6 and the two unknown serotypes, are most closely related to the sequence of CVB6. Sequences among these isolates varied from 82.3 to 99% in the 5′NTR and from 69 to 99% in VP1, indicating no cross contamination. The pathogenicity of these viruses in adult and suckling mice was assessed. None caused pathologic changes in the hearts of adult MF-1 or SWR mice, although pancreatitis was evident. However, the four CVB6-like viruses caused death in suckling mice, similar to a virulent coxsackievirus group B3 laboratory strain. In conclusion, the sequence data confirm that coxsackievirus group B serotypes are predominant in the region in which KD is endemic and may be the etiological agents in outbreaks of myocarditis. VP1 genotyping of enteroviruses is accurate and reliable. Animal experiments indicate that isolates may differ in pathogenicity.

scholarly journals Genomic and Phylogenetic Analysis of Hepatitis C Virus Isolates from Argentine Patients: a Six-Year Retrospective Study

2000 ◽  
Vol 38 (12) ◽  
pp. 4560-4568 ◽  
Author(s):  
J. F. Quarleri ◽  
B. H. Robertson ◽  
V. L. Mathet ◽  
M. Feld ◽  
L. Espı́nola ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fragment Length Polymorphism ◽  
Hcv Rna ◽  
Coding Region ◽  
Serum Samples ◽  
Phylogenetic Relatedness ◽  
Clade 1 ◽  
Virus Isolates ◽  
Rflp Typing

Typing of hepatitis C virus (HCV) isolates from Argentine patients was performed by using different methodologies in a population of 243 patients. HCV subtype was assigned based upon restriction fragment length polymorphism (RFLP). HCV RNA genomes obtained from serum samples were classified as belonging to clade 1 (53.5%), 2 (23.0%), or 3 (8.6%); 14.8% of samples showed HCV mixed infections, more frequently implying different subtypes within the same clade. In addition to RFLP typing, phylogenetic relatedness among sequences from both 5′ untranslated region (n = 50) and nonstructural 5B coding region (n = 15) was established.

scholarly journals Molecular strategy for ‘serotyping’ of human enteroviruses

2001 ◽  
Vol 82 (1) ◽  
pp. 79-91 ◽  
Author(s):  
Valérie Caro ◽  
Sophie Guillot ◽  
Francis Delpeyroux ◽  
Radu Crainic
Keyword(s):  
Molecular Typing ◽  
Phylogenetic Trees ◽  
Field Isolate ◽  
Human Enterovirus ◽  
Specific Information ◽  
Genomic Fragment ◽  
Coding Region ◽  
Typing Method ◽  
Genome Variability ◽  
Molecular Typing Method

To explore further the phylogenetic relationships between human enteroviruses and to develop new diagnostic approaches, we designed a pair of generic primers in order to study a 1452 bp genomic fragment (relative to the poliovirus Mahoney genome), including the 3′ end of the VP1-coding region, the 2A- and 2B-coding regions, and the 5′ moiety of the 2C-coding region. Fifty-nine of the 64 prototype strains and 45 field isolates of various origins, involving 21 serotypes and 6 strains untypeable by standard immunological techniques, were successfully amplified with these primers. By determining the nucleotide sequence of the genomic fragment encoding the C-terminal third of the VP1 capsid protein we developed a molecular typing method based on RT–PCR and sequencing. If field isolate sequences were compared to human enterovirus VP1 sequences available in databases, nucleotide identity score was, in each case, highest with the homotypic prototype (74.8 to 89.4%). Phylogenetic trees were generated from alignments of partial VP1 sequences with several phylogeny algorithms. In all cases, the new classification of enteroviruses into five identified species was confirmed and strains of the same serotype were always monophyletic. Analysis of the results confirmed that the 3′ third of the VP1-coding sequence contains serotype-specific information and can be used as the basis of an effective and rapid molecular typing method. Furthermore, the amplification of such a long genomic fragment, including non-structural regions, is straightforward and could be used to investigate genome variability and to identify recombination breakpoints or specific attributes of pathogenicity.

scholarly journals Analysis of methicillin-resistant and methicillin-susceptibleStaphylococcus aureusby a molecular typing method based on coagulase gene polymorphisms

1995 ◽  
Vol 115 (3) ◽  
pp. 419-426 ◽  
Author(s):  
N. Kobayashi ◽  
K. Taniguchi ◽  
K. Kojima ◽  
S. Urasawa ◽  
N. Uehara ◽  
...  
Keyword(s):  
Molecular Typing ◽  
Gene Polymorphisms ◽  
Fragment Length Polymorphism ◽  
Restriction Enzyme Digestion ◽  
Rflp Pattern ◽  
Typing Method ◽  
Methicillin Resistant ◽  
Serological Typing ◽  
Molecular Typing Method ◽  
Rflp Typing

SummaryA molecular typing method forStaphylococcus aureusbased on coagulase gene polymorphisms (coagulase gene typing) was evaluated by examining a total of 240 isolates which comprised 210 methicillin-resistantS. aureus(MRSA) and 30 methicillin-susceptibleS. aureus(MSSA) collected from a single hospital. ByAlulrestriction enzyme digestion of the PCR-amplified 3′-end region of the coagulase gene including 81-bp repeated units, the MRSA and MSSA isolates examined were divided into 6 and 12 restriction fragment length polymorphism (RFLP) patterns, respectively, whereas five patterns were commonly detected in MRSA and MSSA. MRSA isolates that showed a particular RFLP pattern were considered to be predominant in the hospital. Coagulase typing with type-specific antisera was also performed for allS. aureusisolates for comparison. Coagulase types II and VII were most frequently detected and included isolates with four and five differentAluIRFLP patterns, respectively, whereas each of the other coagulase types corresponded to a single RFLP pattern. These results indicated that RFLP typing was more discriminatory than serological typing, for typingS. aureusand demonstrated its utility in epidemiologic investigation ofS. aureusinfection in hospitals.

scholarly journals Mutational Analysis of Mucopolysaccharidosis in Iranian Patients

2021 ◽  
Vol 23 (3) ◽  
Author(s):  
Seyed Hoseinali Saberi ◽  
Shahla Farshidi ◽  
Behnam Kamalidehghan ◽  
Roshanak Jazayeri ◽  
Massoud Houshmand
Keyword(s):  
Genetic Analysis ◽  
Metabolic Disorder ◽  
Sanger Sequencing ◽  
Clinical Characteristics ◽  
Mutational Analysis ◽  
Coding Region ◽  
Sequencing Method ◽  
The World ◽  
Definition Of ◽  
Iranian Patients

: Mucopolysaccharidosis (MPS) is a rare and heterogeneous metabolic disorder with wide phenotypic distributions throughout the world. This study aimed to determine the genetic polymorphisms, contributing to the most common types of MPS in 19 unrelated Iranian patients. The sequence of the coding region and exon‐intron boundaries of the MPS genes were analyzed by Sanger sequencing method. We used the biochemical and clinical characteristics of MPS subjects for genetic analysis. A novel IDUA variant (c.99T>C, p.H33H), a novel nonsense change (c.514C>T, p.R172*) in exon 5 of IDS gene, c.74G > A (p. p.R24H) in SGSH gene, and three variants including (c.607C>T (p.R203*), c.259G>C (p.A87P), and c.683G>A (p.R228Q)) in NAGLU (n-acetyl-alpha-glucosaminidase) were predicted as novel pathogenic mutations. In conclusion, this study broadened genotypic spectrum of Iranian MPS patients, facilitating the definition of disease-associated mutations, which help to provide a more effective approach in MPS carrier detection.

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