title: Digital PCR assay detection of circulating Mycobacterium tuberculosis DNA in pulmonary tuberculosis patient plasma.
abstract: Nucleic acid amplification tests are a major diagnostic tool for pulmonary tuberculosis (PTB). Recently, digital PCR (dPCR) assay has improved sensitivity for the detection of small copy numbers of target molecules. The aim of this study is to explore the utility of dPCR for detecting Mycobacterium tuberculosis (MTB) DNA in PTB patient plasma. Total DNA was purified from plasma samples of newly diagnosed sputum smear-positive PTB patients. Copy numbers of MTB-specific genes in the samples were quantified with dPCR assays targeted for IS6110 or gyrB. A total of 33 PTB patients were enrolled. Significant differences between PTB patients and controls were observed in copy numbers of both targets: IS6110 mean ± SD, 144.8 ± 538.3 vs 0.44 ± 0.49 (copies/20 μL, p = 0.004; Mann-Whitney U test) and gyrB mean ± SD, 359.0 ± 2116 vs 0.07 ± 0.28 (copies/20 μL, p = 0.011; Mann-Whitney U test), respectively. This test had sensitivities of 65% or 29% and a specificity of 93% or 100% with the IS6110-targeted or gyrB-targeted assays, respectively. A dPCR assay successfully detected MTB DNA in PTB patient plasma. This minimally invasive and accurate method has potential to become an alternative diagnostic option.
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Ushio
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan. Electronic address: masakiy@yokohama-cu.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nakashima
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Nagai
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Shibata
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Tashiro
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Toshinori
authors.initials: T
authors.lastname: Tsukahara
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Nagakura
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Horita
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.affiliation: Respiratory Disease Center, Yokohama City University Medical Center, Yokohama, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Shinkai
authors.affiliation: Respiratory Disease Center, Yokohama City University Medical Center, Yokohama, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kudo
authors.affiliation: Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Atsuhisa
authors.initials: A
authors.lastname: Ueda
authors.affiliation: Department of Pulmonology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kaneko
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016022:Case-Control Studies; D056915:DNA Copy Number Variations; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000097:blood*; D005260:Female; D018628:Gene Dosage; D005819:Genetic Markers; D006801:Humans; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D014397:Tuberculosis, Pulmonary / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D055815:Young Adult
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers; D027081:DNA Gyrase
keywords: Circulating DNA; Digital PCR; IS6110; Plasma; Tuberculosis; gyrB