Development of the loop-mediated isothermal amplification method for rapid detection of cytomegalovirus DNA.
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.
Suzuki
Ryota
R
Division of Pharmacy, Fujita Health University Hospital, Toyoake, Aichi, Japan.
Yoshikawa
Tetsushi
T
Ihira
Masaru
M
Enomoto
Yoshihiko
Y
Inagaki
Shoji
S
Matsumoto
Koichi
K
Kato
Koji
K
Kudo
Kazuko
K
Kojima
Seiji
S
Asano
Yoshizo
Y
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
D004279:DNA, Viral