A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

Wongboonma, Wanwisa; Thongnoppakhun, Wanna; Auewarakul, Chirayu

doi:10.1186/1756-8722-4-7

Cited by 17 publications

(4 citation statements)

References 35 publications

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“…AS-PCR has been widely used to detect the gene mutations [ 30 ]. This method relies on specific PCR primers to discriminate wild type and mutant alleles.…”

Section: Resultsmentioning

confidence: 99%

Development of a highly sensitive method for detection of JAK2V617F

2011

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BackgroundPh- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage. JAK2V617F, an activation mutation form of tyrosine kinase JAK2, is found in the majority of patients with MPNs. Studies have demonstrated that JAK2V617F can cause MPNs, and various methods have been developed to detect JAK2V617F for diagnostic purposes. However, a highly sensitive method is still needed for the earliest possible detection and for disease prevention and treatment.MethodsIn the present study, we developed a method dubbed restriction fragment nested allele-specific PCR (RFN-AS-PCR). The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the JAK2V617F mutation site, 2) digestion of the PCR products with restriction enzyme BsaXI which only cleaves the wild type allele, and 3) detection of JAK2V617F by allele-specific PCR with nested primers.ResultsWe tested the sensitivity of the method by using purified plasmid DNAs and blood cell DNAs containing known proportions of JAK2V617F. We were able to detect JAK2V617F with a sensitivity of 0.001%. We further analyzed blood cell DNA samples from 105 healthy donors with normal blood cell counts and found three JAK2V617F-positive cases, which would have remained undetected using a less sensitive method.ConclusionsWe have developed a highly sensitive method that will allow for detection of JAK2V617F at a very early stage. This method may have major implications in diagnosis and prevention of MPNs and related diseases.

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“…AS-PCR has been widely used to detect the gene mutations [ 30 ]. This method relies on specific PCR primers to discriminate wild type and mutant alleles.…”

Section: Resultsmentioning

confidence: 99%

Development of a highly sensitive method for detection of JAK2V617F

2011

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“…Allele specific-PCR (AS-PCR) for detection of T315I mutation in imatinib resistant CML patient's samples was performed using previously described PCR primer sequences [13].…”

Section: Allele Specific-pcr (As-pcr) To Detect T315i Mutationmentioning

confidence: 99%

Promoter Hypermethylation of ATG16L2, TFAP2A, EBF2, Calcitonin, ABL1 Kinase Domain T315I Mutation Association with Imatinib Therapy Resistance and Median Survival in CML Patients of North-East India

Sarma

Kataki

Kumar

et al. 2023

Asian Pac J Cancer Care

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Background and Objectives: Chronic myeloid leukemia (CML) initiation and progression is regulated by epigenetic and genetic alterations. Imatinib therapy resistance in CML patients is important clinical issue. To understand association of kinase domain mutation and promoter hypermethylation of genes with imatinib therapy resistance hold significance in CML patients of North-East India. This study is a hospital based cross sectional study. Methods: A total of Sixty three (n=63) CML patients undergone imatinib mesylate were enrolled for the study. ABL kinase domain T315I mutation was analyzed by Allele specific- PCR (AS-PCR) and confirmed by sequencing. Promoter hypermethylation was analyzed by Methylation specific PCR (MS-PCR). The Chi square test and Fisher exact test used in SPSS ver19. Results: Tyrosine Kinase domain mutation T315I was found in 30.1% (19/63). The promoter hypermethylation of Calcitonin, ATG16L2, TFAP2A, EBF2 gene was detected in 42.9% (n=27), 28.6% (n=18), 38.1% (n=24), 27% (n= 17) CML patients respectively. Median relapse free survival was 21 months and statistically significant for CML patients without T315I mutation compared to patients with T315I mutation who has 12 months median relapse free survival (p=0.005). Median survival was 19 months for patients without EBF2 promoter hypermethylation and also statistically significant compared to CML patients with promoter hypermethylation (12 months) (p=0.026). Interpretation and Conclusions: We conclude that among imatinib resistant CML patients of North- East India harbouring T315I mutation of ABL1 Kinase domain and promoter hypermethylation of EBF2 gene have significantly lower median relapse free survival.

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“…We introduced it to isothermal nucleic acid amplification and constructed an isothermal ARMS (termed iARMS) assay to identify mutation-carried fungicide-resistant crop fungal pathogens. 27 In addition, CRISPR-Cas12a was designed to specifically detect RPA amplicons. 28 The cascade recognition processes ensured a high specificity to discriminate single-nucleotide mutations in the fungal genome.…”

Section: ■ Introductionmentioning

confidence: 99%

Rapid and Sensitive Detection of Fungicide-Resistant Crop Fungal Pathogens Using an Isothermal Amplification Refractory Mutation System

Song

Zeng

et al. 2023

Anal. Chem.

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Fungicide abuse leads to the emergence of fungicide-resistant fungal pathogens, thus posing a threat to agriculture and food safety. Here, we developed an isothermal amplification refractory mutation system (termed iARMS) allowing us to resolve genetic mutations, enabling rapid, sensitive, and potentially field-applicable detection of fungicide-resistant crop fungal pathogens. iARMS yielded a limit of detection of 25 aM via a cascade signal amplification strategy of recombinase polymerase amplification (RPA) and Cas12a-mediated collateral cleavage at 37 °C within 40 min. Specificity for fungicide-resistant Puccinia striiformis (P. striiformis) detection was guaranteed by RPA primers and the flexible sequence of gRNA. The iARMS assay allowed us to detect as low as 0.1% cyp51-mutated P. striiformis that showed resistance to the demethylase inhibitor (DMI), which was 50 times more sensitive than the sequencing techniques. Thus, it is promising for the discovery of rare fungicide-resistant isolates. We applied iARMS to investigate the emergence of fungicide-resistant P. striiformis in western China and found that its proportion was over 50% in Qinghai, Sichuan, and Xinjiang Province. iARMS can serve as a molecular diagnostic tool for crop diseases and facilitate precision plant disease management.

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A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients

Cited by 17 publications

References 35 publications

Development of a highly sensitive method for detection of JAK2V617F

Development of a highly sensitive method for detection of JAK2V617F

Promoter Hypermethylation of ATG16L2, TFAP2A, EBF2, Calcitonin, ABL1 Kinase Domain T315I Mutation Association with Imatinib Therapy Resistance and Median Survival in CML Patients of North-East India

Rapid and Sensitive Detection of Fungicide-Resistant Crop Fungal Pathogens Using an Isothermal Amplification Refractory Mutation System

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