scholarly journals Ποσοτικοποίηση των στελεχών του ιού της ηπατίτιδας Β (HBV) με τις μεταλλαγές που προσδίδουν αντοχή στην αντιϊκή θεραπεία

2011 ◽  
Author(s):  
Φωτεινή Ντζιώρα
Keyword(s):  
Deep Sequencing ◽  
Molecular Beacon ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Mutation System

ΕΙΣΑΓΩΓΗ: Η ανάπτυξη αντοχής στην αντιϊκή θεραπεία έχει σχετισθεί με την εμφάνιση μεταλλαγών και αποτελεί ένα βασικό πρόβλημα στην αντιμετώπιση της λοίμωξης του ιού της ηπατίτιδας Β (HBV). Οι συμβατικές μέθοδοι αδυνατούν να ανιχνεύσουν ιϊκούς υποπληθυσμούς με τις μεταλλαγές αντοχής αν αντιπροσωπεύουν λιγότερο από το 20% του συνολικού πληθυσμού του ιού στο υπό εξέταση δείγμα. ΣΚΟΠΟΣ: Η δημιουργία μιας μεθόδου για την ποσοτικοποίηση των ιϊκών υποπληθυσμών του HBV με τις κυριότερες μεταλλαγές που προσδίδουν αντοχή στην αντιϊκή θεραπεία (V173L, L180M, A181V, T184A, M204I/V και N236T).ΥΛΙΚΟ-ΜΕΘΟΔΟΣ: Η μεθοδολογία του συστήματος αναστολής της γονιδιακής επέκτασης του μεταλλαγμένου τύπου με PCR πραγματικού χρόνου (amplification refractory mutation system, ARMS RT-PCR) συνδυάστηκε με την τεχνολογία του μοριακού φάρου (molecular beacon) χρησιμοποιώντας το σύστημα του LightCycler®. ΑΠΟΤΕΛΕΣΜΑΤΑ: Η μέθοδος ARMS rt-PCR με μοριακό φάρο έχει ευαισθησία 10 αντιγράφων ανά αντίδραση και αναλυτική ευαισθησία 95% για το μεταλλαγμένο τύπο και 100% για το φυσικό τύπο. Η διακριτική ικανότητα της μεθόδου είναι 0.01%-0.25%. Ιϊκοί υποπληθυσμοί με τις μεταλλαγές αντοχής του HBV V173L, L180M, A181V, T184A, M204I και N236T βρέθηκαν σε υψηλή συχνότητα (38/60, 63.3%), σε δείγματα ασθενών με ηπατίτιδα Β, που δεν είχαν λάβει ποτέ θεραπεία με νουκλεοσ(τ)ιδικά ανάλογα. Σε 41 δείγματα ασθενών με HBV λοίμωξη, που είχαν λάβει θεραπεία, εκτός από τις μεταλλαγές αντοχής με επικρατούντα ιϊκό υποπληθυσμό, ανιχνεύθηκαν και ιϊκοί υποπληθυσμοί με μικρότερους λόγους, που πιθανόν να είχαν συμβάλλει στη συνολική εκδήλωση αντοχής για τον κάθε ασθενή.ΣΥΜΠΕΡΑΣΜΑΤΑ: Η μέθοδος ΑRMS rt-PCR με μοριακό φάρο παρουσιάζει συγκρίσιμη ευαισθησία, πιστότητα και διακριτική ικανότητα με άλλες εξαιρετικά ευαίσθητες μοριακές μεθόδους, όπως είναι το ultra-deep sequencing, αλλά πλεονεκτεί σε θέματα κόστους για την ανίχνευση των ιϊκών υποπληθυσμών του ιού της ηπατίτιδας Β με τις κυριότερες μεταλλαγές αντοχής.

scholarly journals Novel manifestations of Warburg micro syndrome type 1 caused by a new splicing variant of RAB3GAP1: a case report

BMC Neurology ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Raziyeh Khalesi ◽  
Ehsan Razmara ◽  
Golareh Asgaritarghi ◽  
Ali Reza Tavasoli ◽  
Yasser Riazalhosseini ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Cerebral White Matter ◽  
Global Developmental Delay ◽  
Spastic Diplegia ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Mutation System ◽  
Warburg Micro Syndrome

Abstract Background The present study aimed to determine the underlying genetic factors causing the possible Warburg micro syndrome (WARBM) phenotype in two Iranian patients. Case presentation A 5-year-old female and a 4.5-year-old male were referred due to microcephaly, global developmental delay, and dysmorphic features. After doing neuroimaging and clinical examinations, due to the heterogeneity of neurodevelopmental disorders, we subjected 7 family members to whole-exome sequencing. Three candidate variants were confirmed by Sanger sequencing and allele frequency of each variant was also determined in 300 healthy ethnically matched people using the tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. To show the splicing effects, reverse transcription-PCR (RT-PCR) and RT-qPCR were performed, followed by Sanger sequencing. A novel homozygous variant—NM_012233.2: c.151-5 T > G; p.(Gly51IlefsTer15)—in the RAB3GAP1 gene was identified as the most likely disease-causing variant. RT-PCR/RT-qPCR showed that this variant can activate a cryptic site of splicing in intron 3, changing the splicing and gene expression processes. We also identified some novel manifestations in association with WARBM type 1 to touch upon abnormal philtrum, prominent antitragus, downturned corners of the mouth, malaligned teeth, scrotal hypoplasia, low anterior hairline, hypertrichosis of upper back, spastic diplegia to quadriplegia, and cerebral white matter signal changes. Conclusions Due to the common phenotypes between WARBMs and Martsolf syndrome (MIM: 212720), we suggest using the “RABopathies” term that can in turn cover a broad range of manifestations. This study can per se increase the genotype-phenotype spectrum of WARBM type 1.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

scholarly journals The genotyping of Infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction

2014 ◽  
Vol 26 (6) ◽  
pp. 721-733 ◽  
Author(s):  
Shr-Wei Huang ◽  
Chia-Fang Ho ◽  
Kun-Wei Chan ◽  
Min-Chung Cheng ◽  
Jui-Hung Shien ◽  
...  
Keyword(s):  
Amplification Refractory Mutation System ◽  
Wild Type ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Infectious Bronchitis ◽  
Melting Temperatures ◽  
Qrt Pcr ◽  
Multiplex Amplification ◽  
Mutation System ◽  
Polymerase Chain

Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China.

scholarly journals Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique

2019 ◽  
Vol 11 (3) ◽  
Author(s):  
Narutchala Suwannakhon ◽  
Tanapat Pangeson ◽  
Teerapat Seeratanachot ◽  
Khwanruedee Mahingsa ◽  
Arunee Pingyod ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Beta Thalassemia ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Mutation System ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Thalassemia Mutation

We propose using a modified amplification refractory mutation system real-time polymerase chain reaction (ARMS RTPCR) technique to exclude the invasive prenatal diagnosis for a non-paternally inherited beta thalassemia mutation in couples atrisk for having a baby with CHBT. The ARMS RT-PCR method was performed for 36 at-risk couples by using isolated fetal cell-free DNA from maternal plasma. The modified ARMS RT-PCR primers targeted one of the following paternally inherited beta thalassemia mutation: -28 A→G, CD17 A→T, CD 26 G→A, IVS1-1 G→T and CD 41-42 -CTTT. The method could be successfully employed for NIPST starting with the 7th week of gestation. The results showed that 19 pregnant women were negative for PIBTM (53%). After an on-track and on-time of one year, including postnatal thalassemia blood tests, none of the babies showed symptoms or signs of beta thalassemia disease. We concluded that the modified ARMS RT-PCR method was an accurate, cost-effective and feasible method for use as a NIPST for at-risk couples with the potential of having a baby with CHBT.

Genetic Variation in TNF-α, its Relation with Inflammatory Biomarkers and Susceptibility to Rheumatoid Arthritis

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Khadiga Ahmed Ismail
Keyword(s):  
Rheumatoid Arthritis ◽  
Serum Level ◽  
Functional Disability ◽  
Serum Levels ◽  
Amplification Refractory Mutation System ◽  
Reactive Protein ◽  
Tnf Α ◽  
Mutation System ◽  
Potential Factor ◽  
Factor Α

Background: Tumor necrosis Factor-α (TNF-α) is encoded and controlled by TNF-α gene, which is involved in rheumatoid arthritis (RA) susceptibility. This research aimed to identify genetic variations of TNF-α (G308A) and to establish its association with inflammatory markers in Rheumatoid Arthritis predisposition. Methods: In the present study, fifty RA patients and fifty volunteers were involved and evaluated for the C-reactive protein, rheumatoid factor, and TNF-α were estimated by ELISA, Erythrocyte Sedimentation Rate (ESR) by Wintergreen method and for TNF-α-308 G>A polymorphism by polymerase chain reaction with amplification refractory mutation system (PCR-ARMS). Results: The CRP, RF, ESR and TNF-α were significantly elevated in RA patients relative to controls. The serum level TNF-α was also significantly elevated in female patients and in patients ≥50 years. Analysis of TNF-308 gene polymorphism revealed that GG genotypes were more prevalent in RA patients than in the healthy individuals and that GG genotype may be a potential factor to RA. The G allele was more common in RA than in the control. Elevated TNF-α serum levels were significantly associated the GG genotype and functional disability in RA patients. Conclusion: TNF-α promoter 308polymorphism GG genotype may be considered as a risk factor for RA and the TNF-α serum level was significantly related to the functional disability in the disease.

Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency in the Shenzhen Population

2021 ◽  
pp. 1-7
Author(s):  
Jian Gao ◽  
Sheng Lin ◽  
Shiguo Chen ◽  
Qunyan Wu ◽  
Kaifeng Zheng ◽  
...  
Keyword(s):  
G6pd Deficiency ◽  
Amplification Refractory Mutation System ◽  
Gene Variants ◽  
Coding Region ◽  
G6pd Gene ◽  
Mutation System ◽  
Hotspot Mutations ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Generation Sequencing

<b><i>Background:</i></b> Glucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. This study aimed to characterize the G6PD gene variant distribution in Shenzhen of Guangdong province. <b><i>Methods:</i></b> A total of 33,562 individuals were selected at the hospital for retrospective analysis, of which 1,213 cases with enzymatic activity-confirmed G6PD deficiency were screened for G6PD gene variants. Amplification refractory mutation system PCR was first used to screen the 6 dominant mutants in the Chinese population (c.1376G&#x3e;T, c.1388G&#x3e;A, c.95A&#x3e;G, c.1024C&#x3e;T, c.392G&#x3e;T, and c.871G&#x3e;A). If the 6 hotspot variants were not found, next-generation sequencing was then performed. Finally, Sanger sequencing was used to verify all the mutations. <b><i>Results:</i></b> The incidence of G6PD deficiency in this study was 3.54%. A total of 26 kinds of mutants were found in the coding region, except for c.-8-624T&#x3e;C, which was in the noncoding region. c.1376G&#x3e;T and c.1388G&#x3e;A, both located in exon 12, were the top 2 mutants, accounting for 68.43% of all individuals. The 6 hotspot mutations had a cumulative proportion of 94.02%. <b><i>Conclusions:</i></b> This study provided detailed characteristics of G6PD gene variants in Shenzhen, and the results would be valuable to enrich the knowledge of G6PD deficiency.

scholarly journals Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

2017 ◽  
Vol 5 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Hemanta Kumari Chaudhary ◽  
Mitesh Shrestha ◽  
Prakash Chaudhary ◽  
Bal Hari Poudel
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rpob Gene ◽  
Amplification Refractory Mutation System ◽  
Chain Reaction ◽  
Arms Pcr ◽  
Mutation System ◽  
Mdr Tb ◽  
Total Dna ◽  
Polymerase Chain ◽  
Rifampin Resistance

Multidrug-resistant tuberculosis (MDR-TB) has become a serious worldwide threat including in Nepal. MDR-TB refers to the pathological condition whereby Mycobacterium tuberculosis becomes resistant to the first line of drug treatment i.e. rifampin and isoniazid. Resistance to rifampin (RIF) is mainly caused by the mutations in the rpoB gene which codes for the β-subunit of RNA polymerase. In this study, Amplification Refractory Mutation System – Polymerase Chain Reaction (ARMS – PCR) technique has been used to detect mutations in the rpoB gene of Mycobacterium tuberculosis. Total DNA samples of 34 phenotypic MDR-TB were subjected to ARMS – PCR using three different codon specific primers (516, 526 and 531). These three codons occupy large portion of total mutation responsible for rifampin resistance. Out of the total DNA samples, all were bearing mutation in at least one of the three codons mentioned. Of those bearing mutation, the highest number had mutation in codon 531 (97.05 %) followed by codon 516 (17.64 %) and finally in codon 526 (11.76%) respectively. Hence, ARMS – PCR may be used as an alternative diagnostic technique for detection of rifampin resistance in Mycobacterium tuberculosis strains, especially for a developing country like Nepal.Int. J. Appl. Sci. Biotechnol. Vol 5(1): 81-85

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