Journal of clinical laboratory analysis · 2015
Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay for Quantitative Detection of BCR/ABL Fusion Gene in Chronic Myelogenous Leukemia.
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Manthawornsiri Yanaphat, Polpanich Duangporn, Yamkamon Vichanan, Thiramanas Raweewan, Hongeng Suradej, Rerkamnuaychoke Budsaba, Jootar Saengsuree, Tangboriboonrat Pramuan, Jangpatarapongsa Kulachart
Аннотация
BACKGROUND: Magnetic nanoparticles (MNPs) have been widely used in medical diagnostic research. In this work, two technologies, MNPs and polymerase chain reaction (PCR), were combined to increase detection sensitivity and specificity. A novel technique based on the MNPs-PCR enzyme-linked gene assay (MELGA) was developed for detection of the BCR/ABL abnormal gene in chronic myelogenous leukemia (CML) patients.
METHODS: An MNPs-labeled BCR forward primer and a biotin-labeled ABL reverse primer were used to specifically amplify the target gene. After magnetic separation, the PCR product bound to MNPs labeled with streptavidin-conjugated horseradish peroxidase was incubated with the peroxidase substrate and hydrogen peroxide to generate the colorimetric signal.
RESULTS: When compared with real-time quantitative-PCR (RQ-PCR), the MELGA technique exhibited an increased sensitivity of <1 fg with high specificity for the BCR/ABL fusion gene in CML patients. In addition, MELGA colorimetric results correlated well with the number of copies obtained from RQ-PCR.
CONCLUSION: This simple and cost-effective technique is suitable for monitoring CML patients during targeted therapy (tyrosine kinase inhibitors) especially in rural hospitals.
METHODS: An MNPs-labeled BCR forward primer and a biotin-labeled ABL reverse primer were used to specifically amplify the target gene. After magnetic separation, the PCR product bound to MNPs labeled with streptavidin-conjugated horseradish peroxidase was incubated with the peroxidase substrate and hydrogen peroxide to generate the colorimetric signal.
RESULTS: When compared with real-time quantitative-PCR (RQ-PCR), the MELGA technique exhibited an increased sensitivity of <1 fg with high specificity for the BCR/ABL fusion gene in CML patients. In addition, MELGA colorimetric results correlated well with the number of copies obtained from RQ-PCR.
CONCLUSION: This simple and cost-effective technique is suitable for monitoring CML patients during targeted therapy (tyrosine kinase inhibitors) especially in rural hospitals.