title: Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.
abstract: BACKGROUND
  Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate.
  
  
  METHODS
  We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples.
  
  
  RESULTS
  Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR.
  
  
  CONCLUSIONS
  We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.
authors.affiliation: Clinical Laboratory Center, Toyama University Hospital, Toyama, 930-0194, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Clinical Laboratory Center, Toyama University Hospital, Toyama, 930-0194, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Niimi
authors.affiliation: Department of Obstetrics & Gynecology, Toyama University Hospital, Toyama, 930-0194, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Yoneda
authors.affiliation: Department of Obstetrics & Gynecology, Toyama University Hospital, Toyama, 930-0194, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yoneda
authors.affiliation: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa, 921-8836, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Mori
authors.affiliation: Life Science Center, Hokkaido Mitsui Chemicals, Inc., Hokkaido, 073-0138, Japan.
authors.forename: Homare
authors.initials: H
authors.lastname: Tabata
authors.affiliation: Life Science Center, Hokkaido Mitsui Chemicals, Inc., Hokkaido, 073-0138, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Minami
authors.affiliation: Department of Obstetrics & Gynecology, Toyama University Hospital, Toyama, 930-0194, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Saito
authors.affiliation: Clinical Laboratory Center, Toyama University Hospital, Toyama, 930-0194, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Kitajima
mesh_terms: D000293:Adolescent; D000328:Adult; D000653:Amniotic Fluid / Q000382:microbiology*; D017931:DNA Primers / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004795:Enzyme Stability; D005057:Eukaryotic Cells / Q000378:metabolism*; D005260:Female; D005658:Fungi / Q000302:isolation & purification; D006801:Humans; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification*; D007752:Obstetric Labor, Premature / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013696:Temperature; D014509:Ureaplasma / Q000235:genetics / Q000302:isolation & purification*; D055815:Young Adult
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D004259:DNA-Directed DNA Polymerase