title: Development of the loop-mediated isothermal amplification method for rapid detection of cytomegalovirus DNA.
abstract: Cytomegalovirus (CMV) loop-mediated isothermal amplification (LAMP) was performed on DNA extracted from CMV (AD-169)-, herpes simplex virus (HSV) 1 (KOS)-, HSV-2 (186)-, varicella-zoster virus (Oka-vaccine)-, human herpesvirus (HHV)-6 A (U1102)-, HHV-6 B (Z29)-, and HHV-7 (RK)-infected cells. Although amplified CMV demonstrated typical ladder patterns, no LAMP product was detected in reactions performed with other viral DNAs. The sensitivity of the CMV LAMP was 500 copies/tube, as determined by either agarose gel electrophoresis or turbidity assay. To determine whether CMV LAMP could be used for quantitative analysis of viral DNA, threshold times, defined as the time (in seconds) to reach the threshold level (0.1), were measured by amplification of serial dilutions of the plasmid DNA. The standard curve exhibited a correlation coefficient of 0.944, a slope of -208.1, and a y-intercept of 3261.4. Following these initial validation experiments, we analyzed 180 samples collected serially from 20 pediatric hematopoietic stem cell transplant recipients. Detection of CMV DNA in whole blood (WB) was tested by CMV LAMP and real-time polymerase chain reaction (PCR). When >500 copies/tube (>5000 copies/200 microl of WB) was defined as positive for CMV infection, the sensitivity, specificity, positive predictive value, and negative predictive values of the CMV LAMP were 80.0, 98.9, 66.7, and 99.4%, respectively.
authors.affiliation: Division of Pharmacy, Fujita Health University Hospital, Toyoake, Aichi, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Suzuki
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Yoshikawa
authors.forename: Masaru
authors.initials: M
authors.lastname: Ihira
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Enomoto
authors.forename: Shoji
authors.initials: S
authors.lastname: Inagaki
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Koji
authors.initials: K
authors.lastname: Kato
authors.forename: Kazuko
authors.initials: K
authors.lastname: Kudo
authors.forename: Seiji
authors.initials: S
authors.lastname: Kojima
authors.forename: Yoshizo
authors.initials: Y
authors.lastname: Asano
mesh_terms: D002648:Child; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000821:virology; D004279:DNA, Viral / Q000032:analysis* / Q000097:blood / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D019337:Hematologic Neoplasms / Q000150:complications; D018380:Hematopoietic Stem Cell Transplantation; D018259:Herpesvirus 1, Human / Q000235:genetics; D018258:Herpesvirus 2, Human / Q000235:genetics; D014645:Herpesvirus 3, Human / Q000235:genetics; D015654:Herpesvirus 6, Human / Q000235:genetics; D016199:Herpesvirus 7, Human / Q000235:genetics; D006801:Humans; D009391:Nephelometry and Turbidimetry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014180:Transplantation
chemical_list: D004279:DNA, Viral