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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Quantitative detection of PML-RARalpha fusion transcript by real-time PCR with a single primer pair
Mariko Takenokuchi1, Yuji Nakamachi, Keiko Yoneda
1Faculty of Pharmacological Science, Himeji Dokkyo University, Himeji, Hyogo 670-8524, Japan. tmariko@himeji-du.ac.jp
Abstract:
Quantitative detection of minimal residual disease has prognostic value for some leukemias. Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARalpha fusion gene from t(15;17). Added to three PML-RARalpha isoforms, alternative spliced forms of PML exons give rise to multiple isoforms even within a single patient. To date, multiple primer pairs for the detection of the various PML-RARalpha transcripts have been designed, potentially generating some nonspecific amplification products. Here, we established a real-time quantitative PCR (RQ-PCR) strategy with a single primer pair using LightCycler (sp-RQ-PCR), which could simultaneously detect three isoforms with equal specificity and sensitivity as well as alternative spliced forms. Results obtained with sp-RQ-PCR for 39 samples from 15 APL patients and 31 non-APL samples were compared with those with TaqMan assay with three primer pairs. In two of the APL samples, PML-RARalpha was detected in the TM, but not in the sp-RQ-PCR or nested PCR. Furthermore, the sp-RQ-PCR showed no positive results for the 31 non-APL samples, whereas the TM identified 13% (4/31) as positive. Electrophoresis detected some artifacts in the TM, which do not correspond to PML-RARalpha. We conclude that our sp-RQ-PCR is specific enough to identify various forms of PML-RARalpha and yields no false-positive results.
Insights
A new single-primer real-time quantitative PCR (RQ-PCR) method accurately detects multiple PML-RARalpha isoforms in acute promyelocytic leukemia (APL). This specific method avoids false positives, improving minimal residual disease monitoring in APL patients.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- Minimal residual disease (MRD) detection is crucial for leukemia prognosis.
- Acute promyelocytic leukemia (APL) is defined by the PML-RARalpha fusion gene.
- Multiple PML-RARalpha isoforms exist due to alternative splicing, complicating detection.
Purpose of the Study:
- To develop a highly specific and sensitive real-time quantitative PCR (RQ-PCR) strategy for detecting various PML-RARalpha isoforms.
- To establish a single-primer RQ-PCR (sp-RQ-PCR) method for simultaneous detection of multiple isoforms.
- To compare the diagnostic accuracy of the new sp-RQ-PCR method against the TaqMan assay.
Main Methods:
- Development of a novel single-primer real-time quantitative PCR (sp-RQ-PCR) assay using LightCycler technology.
- Simultaneous detection of three major PML-RARalpha isoforms and alternative spliced forms.
- Comparative analysis of sp-RQ-PCR with the TaqMan assay using 39 APL patient samples and 31 non-APL samples.
Main Results:
- The sp-RQ-PCR method demonstrated equal specificity and sensitivity for detecting multiple PML-RARalpha isoforms.
- The TaqMan assay produced false-positive results in 13% of non-APL samples and detected artifacts.
- sp-RQ-PCR correctly identified PML-RARalpha in APL samples, showing no false positives in non-APL samples.
Conclusions:
- The developed sp-RQ-PCR strategy is a specific and reliable method for identifying diverse PML-RARalpha transcripts.
- This assay effectively detects minimal residual disease in APL without generating false-positive results.
- sp-RQ-PCR offers an improved approach for accurate diagnosis and monitoring of APL.
