Development and evaluation of a novel multiple-primer PCR amplification refractory mutation system for the rapid detection of mutations conferring rifampicin resistance in codon 425 of the rpoB gene of Mycobacterium leprae

2008 ◽  
Vol 57 (2) ◽  
pp. 179-184 ◽  
Author(s):  
Bishwa Raj Sapkota ◽  
Chaman Ranjit ◽  
Kapil Dev Neupane ◽  
Murdo Macdonald
Keyword(s):  
Rapid Detection ◽  
Pcr Amplification ◽  
Mycobacterium Leprae ◽  
Rifampicin Resistance ◽  
Amplification Refractory Mutation System ◽  
Wild Type ◽  
Specific Primer ◽  
Simple Method ◽  
Mutation System ◽  
Detection Of Mutations

Rifampicin-resistant Mycobacterium leprae is regularly reported and drug resistance is a major threat for the elimination of leprosy. There is an urgent need for a simple method that can detect rifampicin resistance in clinical isolates. This study developed a multiple-primer PCR amplification refractory mutation system, a simple, reliable and economical method for clinical specimens that allowed the rapid detection of mutations in the nucleotides of the codon for Ser425 of the M. leprae rpoB gene, mutation of which to Leu, Met or Phe is associated with rifampicin resistance. The approach involved a multiple-primer PCR in which both mutant-specific and normal sets of primers were included in the reaction. The mutant-specific primer was complementary to the corresponding sequence of the wild-type gene except for one additional deliberate mismatch at the fourth nucleotide from the 3′-OH terminus. A single mismatch has little influence on the yield of PCR products, but if there are two mismatches as a result of mutation at the position being tested, the mutant-specific primer will not function in PCR under appropriate conditions, leading to no yield of PCR product from the mutant allele. The assay was evaluated successfully using a panel of plasmids and M. leprae reference strains carrying the wild-type or known rpoB mutations. The assay was subsequently applied to M. leprae DNA extracts from skin biopsies taken from patients. In all biopsy samples, the wild-type allele was detected for Ser425. The PCR results correlated with rifampicin susceptibility, as also measured by the traditional in vivo mouse footpad technique.

scholarly journals Rapid Detection of blaKPC-9 Allele from Clinical Isolates

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 487
Author(s):  
Konstantina Gartzonika ◽  
Petros Bozidis ◽  
Ephthalia Priavali ◽  
Hercules Sakkas
Keyword(s):  
Pcr Amplification ◽  
Diagnostic Methods ◽  
Amplification Refractory Mutation System ◽  
Gene Variants ◽  
Simple Method ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Geographical Regions ◽  
Mutation System ◽  
Additional Procedure ◽  
Endonuclease Digestion

The emergence of Klebsiella pneumoniae carbapenemase (KPC) nosocomial outbreaks related to specific blaKPC gene variants dictates the need for applicable diagnostic methods for allele discrimination. We report here a simple method of blaKPC-9 allele recognition based on a combination of endonuclease digestion analysis and PCR amplification using unique primers. K. pneumoniae isolates carrying the blaKPC gene were tested. Digestion with RsaI restriction endonuclease was found to efficiently differentiate the blaKPC-2 from the blaKPC-9 variants into two distinct groups of digestion patterns named KPC-2-like and KPC-9-like, respectively. An additional procedure, the amplification refractory mutation system (ARMS) method, was applied to identify the variant within the same group. The principles of this procedure could be developed to identify several blaKPC gene variants, as well as monitoring the spread and evolution of specific KPC variants within local geographical regions.

scholarly journals Association of AFF3 Gene Polymorphism rs10865035 with Rheumatoid Arthritis: A Population-Based Case-Control Study on a Pakistani Cohort

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Yasir Ali ◽  
Suleman Khan ◽  
Yangchao Chen ◽  
Nadia Farooqi ◽  
Zia-Ul Islam ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Additive Model ◽  
Pcr Amplification ◽  
Population Based ◽  
Recessive Model ◽  
Taqman Assay ◽  
Amplification Refractory Mutation System ◽  
Multiple Loci ◽  
Geographical Variations ◽  
Mutation System

Rheumatoid arthritis (RA) is one of the complex diseases with the involvement of the genetic as well as environmental factors in its onset and severity. Different genome-wide association and candidate gene studies have shown the role of several genetic variants in multiple loci/genes with ethnical and geographical variations. This study was designed to detect the association of a single-nucleotide polymorphism (SNP) rs10865035 in the AFF3 gene with the genetic background of rheumatoid arthritis (RA) in the Pakistani cohort. A total of 703 individuals, including 409 RA patients and 294 healthy controls, were genotyped using TaqMan assay and Tri primer ARMS-PCR (amplification-refractory mutation system-polymerase chain reaction) methods. The association of rs10865035 with the RA was statistically determined using different models. Interestingly, besides the homozygous recessive model (G/G vs. A/G + A/A) (OR = 1.693(1.06–2.648); P  = 0.025), all other models, which included the codominant (χ2 = 5.169; P  = 0.075), homozygous dominant (A/A vs. G/G + A/G) (OR = 0.867 (0.636–1.187); P  = 0.41), heterozygous (A/G vs. A/A + GG) (OR = 0.491 (0.667–1.215); P  = 0.49), and additive model (OR = 0.826 (0.665–1.027); P  = 0.08) showed insignificant distribution of the genotypes among the cases and controls. These findings suggest that the AFF3 gene (rs10865035) has no significant role in the onset of RA in the Pakistani population.

scholarly journals RAPID DETECTION OF -THALASSEMIA MUTATIONS IN THAILAND USING MULTIPLEX ARMS

2017 ◽  
Vol 25 (2) ◽  
pp. 321-332
Author(s):  
D. Shimbhu ◽  
S. Mirasena ◽  
M. Sanguansermsri ◽  
T. Sanguansermsri
Keyword(s):  
Rapid Detection ◽  
Direct Sequencing ◽  
Medical Personnel ◽  
Amplification Refractory Mutation System ◽  
Single Tube ◽  
Multiplex Amplification ◽  
Mutation System ◽  
Microcolumn Chromatography ◽  
Thalassemia Trait

The number of mutations underlining b-thalassemia generate a wide variety of different clinical phenotypes. An understanding of the genotype is important for medical personnel in order to provide proper counseling to patients and their families. Characterization of these mutations should aid the planning of a prenatal diagnosis program for bthalassemia. The heterogeneity of the mutations makes it difficult and time consuming to identify the mutation in some individuals. We developed a single-tube multiplex amplification refractory mutation system (multiplex ARMS) to identify common ethnic- specific b-thalassemia mutations. Confirmation of multiplex ARMS results was carried out using direct sequencing. Three thousand three hundred twenty two people from Phitsanulok province were screened for the b-thalassemia trait by quantitation of HbA2 with microcolumn chromatography and the genotypes of mutations were characterized using multiplex ARMS and direct sequencing. We found that the deletion at codons 41/42 (-TCTT) was the most frequent (48%), codon 17 (A®T) (30%), -28 (A®G) (6%) and IVS-I-1(G®T) (6%) were the second and third in frequency respectively. A -87 (C®A) mutation (4%), IVS II-654 (C®T) (2%), codons 71/72 (+A) (2%) and codon 35 (C®A) mutations (2%) were also found. These techniques were found to be a valuable tool for analysis of b-thalassemia mutations because they are accurate, simple, and speedy in operation. The application for the diagnosis of severe thalassemia in high-risk pregnancies is promising.    

scholarly journals Molecular Authentication of Dendrobium Species by Multiplex Polymerase Chain Reaction and Amplification Refractory Mutation System Analysis

2012 ◽  
Vol 137 (6) ◽  
pp. 438-444 ◽  
Author(s):  
Chu-Hui Chiang ◽  
Tsong-Ann Yu ◽  
Shu-Fang Lo ◽  
Chao-Lin Kuo ◽  
Wen-Huang Peng ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
System Analysis ◽  
Pcr Amplification ◽  
Amplification Refractory Mutation System ◽  
Chain Reaction ◽  
5.8S Rdna ◽  
Traditional Chinese Herbal Medicine ◽  
Mutation System ◽  
Polymerase Chain

The genus Dendrobium is important in traditional Chinese herbal medicine, and the precise identification of Dendrobium species is critical for the treatment and for pharmacological research. In the present study, a ribosomal DNA (rDNA) internal transcribed spacer (ITS) region-based analysis was used to ascertain the phylogenetic relationship among 20 Dendrobium species. The lengths of the ITS regions among the 20 species ranged from 636 to 653 bp, and the identities of the rDNA regions among the different species ranged from 75.7% to 99.1%. The results also showed that the ITS1 and ITS2 regions exhibit more variation than the 5.8S rDNA. A phylogenetic tree derived from the ITS sequence indicated that six medicinal Dendrobium species, of which five are common medicinal plants in the Taiwan market, were closely related and shared a common clade. Multiplex polymerase chain reaction (PCR) amplification was successfully performed to identify the six medicinal Dendrobium species, and amplification refractory mutation system (ARMS) PCR was used to distinguish D. tosaense specifically from the 19 other Dendrobium species. The established PCR-based (multiplex and ARMS) analyses can be used for the authentication of the raw materials of medicinal Dendrobium from other species.

scholarly journals Analysis of the Chimeric CYP21P/CYP21 Gene in Steroid 21-Hydroxylase Deficiency

2000 ◽  
Vol 46 (5) ◽  
pp. 606-611 ◽  
Author(s):  
Hsien-Hsiung Lee ◽  
Jan-Gowth Chang ◽  
Chang-Hai Tsai ◽  
Fuu-Jen Tsai ◽  
Hsiang-Tai Chao ◽  
...  
Keyword(s):  
Congenital Adrenal Hyperplasia ◽  
Direct Method ◽  
Pcr Amplification ◽  
Chimeric Gene ◽  
Wild Type ◽  
Adrenal Hyperplasia ◽  
Specific Primer ◽  
Hydroxylase Deficiency ◽  
Cyp21 Gene ◽  
21 Hydroxylase Deficiency

Abstract Background: A single nonfunctional chimeric gene with its 5′ and 3′ ends corresponding to CYP21P and CYP21, respectively, is caused by unequal gene crossover in the CYP21 genes during meiosis. The presence of the chimeric CYP21P/CYP21 molecule can not be detected by conventional PCR methods and therefore may be lost in PCR amplification. This leads to a false result and diagnostic discordance. Methods: We developed a rapid and direct method to detect a chimeric CYP21P/CYP21 gene that uses a 3′-specific primer for the CYP21 gene and two different 5′ primers for both CYP21 and CYP21P to amplify the wild-type CYP21 and the chimeric CYP21P/CYP21 genes. A secondary PCR that can differentiate the chimeric from the wild-type gene was also performed. The PCR product was directly analyzed on agarose gel. Results: After careful titration, we found that earlier failure to detect the chimeric CYP21P/CYP21 gene could be caused by unequal concentrations of two independent alleles as the PCR template or by the lack of primers to amplify chimeric molecules. We successfully amplified the chimeric gene using our improved method. Conclusions: The chimeric CYP21P/CYP21 is present in a large portion of congenital adrenal hyperplasia patients. By adding a CYP21P/CYP21-specific primer, we were able to amplify and detect both homozygous and heterozygous chimeric genes. Therefore, our new PCR-based assay is a more effective way to analyze congenital adrenal hyperplasia mutations.

scholarly journals Polimorfismos de un solo nucleótido representativos para los alelos clásicos del antígeno leucocitario humano en familias antioqueñas con diabetes mellitus tipo 1

Biomédica ◽  
2018 ◽  
Vol 38 (3) ◽  
pp. 329-337 ◽  
Author(s):  
Diana Clobeth Sarrazola ◽  
Alejandra Marcela Rodríguez ◽  
Martín Toro ◽  
Alejandra Vélez ◽  
Jorge García-Ramírez ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Pcr Amplification ◽  
Human Leukocyte ◽  
Amplification Refractory Mutation System ◽  
Single Nucleotide ◽  
Leukocyte Antigen ◽  
Tag Snp ◽  
Pcr Rflp ◽  
Mutation System ◽  
Polymerase Chain

Introducción. La región del antígeno leucocitario humano (Human Leukocyte Antigen, HLA) se ha asociado claramente con enfermedades autoinmunitarias, como la diabetes mellitus de tipo 1. Los polimorfismos representativos de un solo nucleótido (tag Single Nucleotide Polymorphism, tag SNP) constituyen una forma alternativa de evaluar los alelos clásicos del HLA. En la población europea se ha reportado un grupo de tag SNP para múltiples alelos clásicos relacionados con la predisposición o la resistencia frente a dicha enfermedad.Objetivo. Validar la metodología basada en los tag SNP enfocada en la inferencia de alelos HLA clásicos, y evaluar su asociación con la diabetes mellitus de tipo 1 en una muestra de familias antioqueñas.Materiales y métodos. Se estudió una muestra de 200 familias antioqueñas con uno a dos hijos afectados por diabetes mellitus de tipo 1. Se genotipificaron 13 SNP mediante el ARMS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) con cuatro iniciadores, o mediante la PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). Además, se evaluó la validez de los tag SNP de 1.000 genomas reportados en europeos en una muestra de 60 individuos de la población colombiana de Medellín. Se hicieron las pruebas de desequilibrio de la transmisión, de desequilibrio de ligamiento y de equilibrio de Hardy-Weinberg.Resultados. En la población de estudio no se encontró suficiente desequilibrio de ligamiento entre los SNP y los alelos clásicos evaluados, por lo cual no fue posible inferir los alelos clásicos del HLA para el conjunto de familias con diabetes mellitus de tipo 1. El estudio de asociación evidenció que esta región aporta factores tanto de riesgo como de protección para el desarrollo de la enfermedad. Los tag SNP apropiados para la muestra de estudio se determinaron usando los SNP ubicados en la región HLA en la base de datos del 1000 Genomes Project en la mencionada población.Conclusiones. Los patrones de desequilibrio de ligamiento en la población estudiada fueron diferentes a los reportados para la población europea. A pesar de esto, se encontró evidencia clara sobre el papel de la región HLA en el riesgo de padecer diabetes mellitus de tipo 1 en la población de estudio.

scholarly journals Rapid visual detection of FecB gene expression in sheep

2020 ◽  
Vol 15 (1) ◽  
pp. 902-911
Author(s):  
Li Liu ◽  
Ruirui Hu ◽  
Cunyuan Li ◽  
Xiaoyue Li ◽  
Wei Ni ◽  
...  
Keyword(s):  
Agricultural Production ◽  
Color Change ◽  
Sybr Green ◽  
Sybr Green I ◽  
Amplification Refractory Mutation System ◽  
Wild Type ◽  
Economic Traits ◽  
Mutation System ◽  
Bright Green ◽  
Polymerase Chain

AbstractSheep play an important role in agricultural production and people’s lives, and fecundity is one of the most important economic traits of sheep for sheep breeders. The Booroola fecundity (FecB) gene has a certain correlation with litter size in sheep. Therefore, this study aims to detect FecB expression quickly, accurately and visually. Here, we used the nucleic acid dye SYBR Green I to detect FecB with the amplification refractory mutation system (ARMS), which can efficiently, rapidly, economically and visually detect FecB expression in sheep. After ARMS polymerase chain reaction (PCR), SYBR Green I was directly added to the ARMS products, and whether the sheep carried FecB was judged by directly observing the color change in the PCR tube. Homozygous (BB) or heterozygous (B+) samples with FecB mutation were bright green, while wild type (++) samples without FecB were orange yellow. This study suggested that this method has 100% accuracy and 0.5 ng/µL sensitivity. To our knowledge, this is the first report that shows the integration of the ARMS with SYBR Green I to detect FecB expression in sheep visually.

scholarly journals Multiplexed Assays for Detection of Mutations in PIK3CA

2008 ◽  
Vol 54 (4) ◽  
pp. 757-760 ◽  
Author(s):  
Ruth E Board ◽  
Nicola J Thelwell ◽  
Paul F Ravetto ◽  
Stephen Little ◽  
Malcolm Ranson ◽  
...  
Keyword(s):  
Pik3ca Mutation ◽  
Clinical Samples ◽  
Amplification Refractory Mutation System ◽  
Pik3ca Mutations ◽  
Specific Pcr ◽  
Mutation System ◽  
Allele Specific ◽  
Phosphoinositide 3 Kinase ◽  
Low Sensitivity ◽  
Detection Of Mutations

Abstract Background: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. Methods: We combined Amplification Refractory Mutation System (ARMS™; AstraZeneca) allele-specific PCR and Scorpions™ (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). Results: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. Conclusions: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.

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