Background: Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RI-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine.
Methods: The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RI-nested PCR using the droplet-PCR machine.
Results: One-step RI-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40 min, which was 10(3)-fold superior to conventional RI nested PCR using three steps in separate tubes. The sensitivity of the one-step RI-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay.
Conclusions: One-step RI-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RI-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories. (C) 2015 Elsevier B.V. All rights reserved.