Clinical application of catalytically cleavable fluorescence probe technology for multiplexing quantification of BCR-ABL1 fusion transcripts.
BACKGROUND
Accurate measurement of BCR-ABL1 fusion transcripts is critical for therapeutic stratification in patients with chronic myelogenous leukemia (CML). Previous studies have reported the variable performance of the existing quantitative reverse transcription polymerase chain reaction (RQ-PCR). Here, we developed a one-step multiplex RQ-PCR method based on the catalytically cleavable fluorescence probe technology for quantification of BCR-ABL1 transcripts.


METHODS
Performance was evaluated with respect to the limit of detection (LoD), linearity, precision, and comparison on the VIIA7 Real-Time PCR system. Multiplex RQ-PCR was performed by the one-step and one-well reaction without the hands-on time.


RESULTS
Our assay showed a LoD of 1.5 pg with linearity in the range of more than 4 logs of dilution. Intraassay, interassay, and total percent CVs at the concentration of 150 ng were 12.8%, 22.6%, and 28.0%, respectively. The assay correlated well with Asuragen's BCR/ABL1 Quant™ kit over a 6 log concentration range (r=0.9967).


CONCLUSIONS
Our assay demonstrated comparable performance characteristics in comparison with previous RQ-PCR based on the TaqMan probe technology.We conclude that our method could be a reliable tool in the clinical setting.
Kyoung-Jin
KJ
Park
Young Min
YM
Woo
Kwangwoo
K
Kim
Seung-Tae
ST
Lee
Chang-Seok
CS
Ki
Hee-Jin
HJ
Kim
Sun-Hee
SH
Kim
Jong-Won
JW
Kim
D055162:Biocatalysis; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D016914:Ribonuclease H / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*
C581634:BCR-ABL1 fusion protein, human; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl; D016914:Ribonuclease H