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.

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.

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 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 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 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 The Story of a Ship Journey, Malaria, and the HBB Gene IVS-II-745 Mutation: Circassian İmmigration to Cyprus

2021 ◽  
Author(s):  
Mahmut C. Ergoren ◽  
Sehime G. Temel ◽  
Gamze Mocan ◽  
Munis Dundar
Keyword(s):  
Autosomal Recessive ◽  
Single Gene ◽  
Pcr Amplification ◽  
Health Condition ◽  
Amplification Refractory Mutation System ◽  
Chain Reaction ◽  
Analysis Technique ◽  
Endemic Malaria ◽  
Mutation System ◽  
Polymerase Chain

Abstract Background During 19th century, the Circassians were secluded from their lands and forced to migrate to Ottoman Empire properties. Approximately 2,346 Circassians were exiled from Istanbul to Cyprus Island. During the deportation journey, many of Circassian were passed away in consequence of malaria and unknown reasons. Overall, 1,351 survivor Circassians managed to reach the island, however, many of them had faced with endemic malaria again in Cyprus. An autosomal recessive hematological disorder thalassemia was the second endemic health condition after malaria, whereas thalassemia carriers show resistance to malaria infections. Materials and Methods A large Cypriot family with 57 members whose grandparents were supposed to be in that ship journey has been investigated in this study. Polymerase chain reaction (PCR)–amplification refractory mutation system (ARMS) analysis technique was used for genotyping the HHB gene. Results The human β-globin (HBB) gene c.316–106C > G (IVS-II-745) (II-745) heterozygous variation have been detected. Conclusion Overall, this study is a very good example for a typical natural selection. In this case, one single gene point mutation did not limit survival in the society; natively, it increased their survival changes to form new colonization and the inheritance of the mutation to the next generations.

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.

Multigene Combined Detection by RT-qPCR Using Cytological Specimens

2021 ◽  
pp. 1-10
Author(s):  
Yang Ma ◽  
Jingxia Zhao ◽  
Yun Du ◽  
Rui Wang ◽  
Xiaokun Ji ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Smoking Status ◽  
Single Gene ◽  
Rna Extraction ◽  
Pcr Amplification ◽  
Driver Mutations ◽  
Amplification Refractory Mutation System ◽  
Driver Genes ◽  
Mutation Status ◽  
Pik3ca Mutations

<b><i>Objective:</i></b> The aim of the study was to investigate the mutation status of multiple driver genes by RT-qPCR and their significance in advanced lung adenocarcinoma using cytological specimens. <b><i>Materials and Methods:</i></b> 155 cytological specimens that had been diagnosed with lung adenocarcinoma in the Fourth Hospital of Hebei Medical University were selected from April to November 2019. The cytological specimens included serous cavity effusion and fine-needle aspiration biopsies. Among cytological specimens, 108 cases were processed by using the cell block method (CBM), and 47 cases were processed by the disposable membrane cell collector method (MCM) before DNA/RNA extraction. Ten drive genes of EGFR, ALK, ROS1, BRAF, KRAS, NRAS, HER2, RET, PIK3CA, and MET were combined detected at one step by the amplification refractory mutation system and ABI 7500 RT-qPCR. <b><i>Results:</i></b> The purity of RNA (<i>p</i> = 0.005) and DNA (<i>p</i> = 0.001) extracted by using the MCM was both significantly higher than that extracted by using the CBM. Forty-seven cases of fresh cell specimens processed by the MCM all succeeded in multigene detections, while of 108 specimens processed by the CBM, 6 cases failed in multigene detections. Among 149 specimens, single-gene mutation rates of EGFR, ALK, ROS1, RET, HER2, MET, KRAS, NRAS, BRAF, and PIK3CA mutations were 57.71%, 6.04%, 3.36%, 2.68%, 2.01%, 2.01%, 1.34%, 0.67%, 0% and 0% respectively, and 6 cases including 2 coexistence mutations. We found that mutation status was correlated with gender (<i>p</i> = 0.047), but not correlated with age (<i>p</i> = 0.141) and smoking status (<i>p</i> = 0.083). We found that the EGFR mutation status was correlated with gender (<i>p</i> = 0.003), age (<i>p</i> = 0.015) and smoking habits (<i>p</i> = 0.007), and ALK mutation status was correlated with age (<i>p</i> = 0.002). <b><i>Conclusion:</i></b> Compared with the CBM, the MCM can improve the efficiency of DNA/RNA extraction and PCR amplification by removing impurities and enriching tumor cells. And we speculate that the successful detection rate of fresh cytological specimens was higher than that of paraffin-embedded specimens. EGFR, ALK, and ROS1 mutations were the main driver mutations in patients with advanced lung adenocarcinoma. We speculate that EGFR and ALK are more prone to concomitant mutations, respectively. Targeted therapies for patients with coexisting mutations need further study.

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