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Loop-Mediated Isothermal Amplification for the Detection of Fusion Gene BCR-ABL1 Expression in Chronic Myeloid Leukaemia Patients

Carlsson Pecharromán, Cecilia (2021) MOBM02 20191
Degree Projects in Molecular Biology
Abstract
A translocation between chromosomes 9 and 22 produces the fusion gene BCR-ABL that leads to the constitutive activity of a tyrosine kinase. This initiates the uncontrolled proliferation of myeloid cells causing Chronic Myeloid Leukaemia (CML). The disorder is treated by tyrosine kinase inhibitors (TKIs) that prevent the progression of CML into more advanced phases. The treatment must be sustained and monitored continuously, which is currently done by quantitative PCR (qPCR). In this paper, we suggest the establishment of Loop-Mediated Isothermal Amplification (LAMP) as the method for detection of fusion gene BCR-ABL1 expression, since LAMP complies with the ideal molecular diagnostic assay. This will be done by comparing the specificity,... (More)
A translocation between chromosomes 9 and 22 produces the fusion gene BCR-ABL that leads to the constitutive activity of a tyrosine kinase. This initiates the uncontrolled proliferation of myeloid cells causing Chronic Myeloid Leukaemia (CML). The disorder is treated by tyrosine kinase inhibitors (TKIs) that prevent the progression of CML into more advanced phases. The treatment must be sustained and monitored continuously, which is currently done by quantitative PCR (qPCR). In this paper, we suggest the establishment of Loop-Mediated Isothermal Amplification (LAMP) as the method for detection of fusion gene BCR-ABL1 expression, since LAMP complies with the ideal molecular diagnostic assay. This will be done by comparing the specificity, sensitivity and selectivity of reverse transcription-LAMP (RT-LAMP) with reverse transcription-PCR (qRT-PCR). (Less)
Popular Abstract
Surveillance of Chronic Myeloid Leukaemia

Chronic Myeloid Leukaemia (CML) is a cancer of the blood cells caused by the fusion of two genes: BCR and ABL1. Most patients are diagnosed in the earliest stage of the disorder when they lack or have mild symptoms. The most common treatments are drugs that prevent the progression of the disorder; however, patients can develop resistance to the prescribed drug. Therefore, it is important to monitor the disorder continuously, to change the treatment if the drug stops working.

The surveillance of CML is currently performed by a technique called quantitative polymerase chain reaction (qPCR). But, we suggested another technique called loop mediated isothermal amplification (LAMP) for this task.... (More)
Surveillance of Chronic Myeloid Leukaemia

Chronic Myeloid Leukaemia (CML) is a cancer of the blood cells caused by the fusion of two genes: BCR and ABL1. Most patients are diagnosed in the earliest stage of the disorder when they lack or have mild symptoms. The most common treatments are drugs that prevent the progression of the disorder; however, patients can develop resistance to the prescribed drug. Therefore, it is important to monitor the disorder continuously, to change the treatment if the drug stops working.

The surveillance of CML is currently performed by a technique called quantitative polymerase chain reaction (qPCR). But, we suggested another technique called loop mediated isothermal amplification (LAMP) for this task. Both techniques consist in the amplification (production of copies) of tiny fragments, in this case products of the fused gene. LAMP can do this at a constant temperature, while qPCR has to change temperatures. This makes LAMP cheaper, faster and easier to use. However, the technique should also be: specific (amplifies only the target), selective (favours one product over another) and sensitive (detects low concentrations).

In order to determine if LAMP could be established as a new technique for the surveillance of CML, we compared the specificity, selectivity and specificity of LAMP with that of qPCR.

Contamination
The results we obtained by amplifying the fusion gene products with LAMP were frequently unclear because the samples were contaminated (other genetic material was combined with our target products). Although, we changed different parameters in the experiment we still had contamination. This makes the results obtained by LAMP unreliable, and because of this we were unable to determine if LAMP or qPCR had better specificity, selectivity and specificity. The next step would be to alter other parameters to observe if those eliminate the contamination.


Masterexamensprojekt i Molekylärbiologi 30 hp 2019
Biologiska institutionen, Lunds universitet

Handledare: Luís Lombardía
Molecular Diagnostics Unit, CNIO (Less)
Please use this url to cite or link to this publication:
author
Carlsson Pecharromán, Cecilia
supervisor
organization
course
MOBM02 20191
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9043923
date added to LUP
2021-05-11 13:17:34
date last changed
2021-05-11 13:17:34
@misc{9043923,
  abstract     = {{A translocation between chromosomes 9 and 22 produces the fusion gene BCR-ABL that leads to the constitutive activity of a tyrosine kinase. This initiates the uncontrolled proliferation of myeloid cells causing Chronic Myeloid Leukaemia (CML). The disorder is treated by tyrosine kinase inhibitors (TKIs) that prevent the progression of CML into more advanced phases. The treatment must be sustained and monitored continuously, which is currently done by quantitative PCR (qPCR). In this paper, we suggest the establishment of Loop-Mediated Isothermal Amplification (LAMP) as the method for detection of fusion gene BCR-ABL1 expression, since LAMP complies with the ideal molecular diagnostic assay. This will be done by comparing the specificity, sensitivity and selectivity of reverse transcription-LAMP (RT-LAMP) with reverse transcription-PCR (qRT-PCR).}},
  author       = {{Carlsson Pecharromán, Cecilia}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Loop-Mediated Isothermal Amplification for the Detection of Fusion Gene BCR-ABL1 Expression in Chronic Myeloid Leukaemia Patients}},
  year         = {{2021}},
}