title: Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.
abstract: 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 RT-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 RT-nested PCR using the droplet-PCR machine.
  
  
  RESULTS
  One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay.
  
  
  CONCLUSIONS
  One-step RT-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 RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories.
authors.affiliation: Graduate School of Science and Technology, Shinshu University, Nagano, Japan; Core Technology Development Center, Seiko Epson Corporation, Suwa, Japan.
authors.forename: Akemi
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan. Electronic address: kmatsuda@shinshu-u.ac.jp.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsuda
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Sueki
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Taira
authors.affiliation: Core Technology Development Center, Seiko Epson Corporation, Suwa, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Uehara
authors.affiliation: Institute of Engineering, Academic Assembly, Shinshu University, Nagano, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Honda
mesh_terms: D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D010316:Particle Size; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D013997:Time Factors
chemical_list: D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl