title: Rapid detection of PML-RARA fusion gene by novel high-speed droplet-reverse transcriptase-polymerase chain reaction: possibility for molecular diagnosis without lagging behind the morphological analyses.
abstract: BACKGROUND
  Acute promyelocytic leukemia (APL) is an aggressive disease requiring prompt diagnosis and treatment. Rapid detection of the PML-RARA fusion gene provides the molecular basis for a highly effective therapy with all-trans retinoic acid. We developed a rapid assay by novel droplet-reverse transcriptase-polymerase chain reaction (droplet-RT-PCR) for the detection of the PML-RARA fusion gene in APL patients.
  
  
  METHODS
  RNA was extracted from 7 samples obtained from 5 APL patients with the PML-RARA fusion gene confirmed by nested RT-PCR and fluorescence in situ hybridization. Using these 7 samples, we evaluated the reaction time and amplification efficiency of the droplet-RT-PCR.
  
  
  RESULTS
  Using the droplet-RT-PCR, we could detect the PML-RARA fusion gene in all 7 samples. The reaction time for 50 cycles of droplet-RT-PCR was 27 min. The amplification by the droplet-RT-PCR assay was considered positive for the PML-RARA fusion gene in less than 22 min, at the point when the fluorescence exceeded the threshold level.
  
  
  CONCLUSIONS
  Our novel droplet-RT-PCR assay is specific for the detection of the PML-RARA fusion gene and has a markedly reduced reaction time. Thus, the novel droplet-RT-PCR assay contributes to the rapid diagnosis of APL without lagging behind the morphological assessment.
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Sueki
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsuda
authors.forename: Chiaki
authors.initials: C
authors.lastname: Taira
authors.forename: Akemi
authors.initials: A
authors.lastname: Yamaguchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Koeda
authors.forename: Fumio
authors.initials: F
authors.lastname: Takagi
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Sugano
authors.forename: Takayuki
authors.initials: T
authors.lastname: Honda
mesh_terms: D000328:Adult; D000368:Aged; D002884:Chromosomes, Human, Pair 15; D002886:Chromosomes, Human, Pair 17; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D015473:Leukemia, Promyelocytic, Acute / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D014178:Translocation, Genetic
chemical_list: D015514:Oncogene Proteins, Fusion; D018168:Receptors, Retinoic Acid; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein