Quantitative real time-polymerase chain reaction method in Bcr-Abl translocation diagnostics.
OBJECTIVE
The quantitative real-time polymerase chain reaction (qRT-PCR) is used in the detection of molecular events involved in leukemogenesis, such as the Bcr-Abl gene translocation, the most important factor in the pathogenesis of chronic myeloid leukaemia (CML). The main aim of our study was to test the reproducibility, specificity and sensitivity of the qRT-PCR in the detection of Bcr-Abl gene translocation.


METHODS
In complementary (c)DNA, isolated from K562 Bcr-Abl positive cell line, we performed qRT-PCR analysis with Bcr-Abl specific primers. For qRT-PCR analysis, we used serial dilutions of the newly synthesized cDNA in order to establish the detection threshold of this method.


RESULTS
Using the specific primers for the Bcr-Abl translocation, we obtained the specific translocation product in cDNA sample of K562 human erythroid leukemia cell line. qRT- PCR showed significant sensitivity with the detection threshold for the Bcr-Abl fluorescent signal, which enabled the precise detection that was accurate within a 10-fold dilution range, and a dynamic range of 5 orders of magnitude.


CONCLUSIONS
The results of our study showed that the application of the qRT-PCR is the optimal method for the detection of Bcr-Abl gene translocation, characterized by high reproducibility, specificity and sensitivity.
Stankov
K
K
Department of Biochemistry, Medical Faculty Novi Sad, University of Novi Sad, Novi Sad, Serbia. stankov_karmen@uns.ac.rs
Mihajlovic
D
D
Stanimirov
B
B
Stankov
S
S
Bajin-Katic
K
K
Mikov
I
I
Popovic
S
S
D000225:Adenine; D000595:Amino Acid Sequence; D001483:Base Sequence; D002892:Chromosomes, Human, Pair 22; D002899:Chromosomes, Human, Pair 9; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics*; D016254:Mutagenesis, Insertional; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014178:Translocation, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D016044:Fusion Proteins, bcr-abl; D000225:Adenine