Single-tube multiplex PCR for rapid and sensitive diagnosis of subgenus B and other subgenera adenoviruses in clinical samples.
We developed a new diagnostic method of subgenus (Sub) B adenovirus (Ad) in clinical samples using non-nested polymerase chain reaction (PCR). Sequences of the conserved hexon-coding region of representative strains of eight serotypes (3, 7, 11, 14, 16, 21, 34 and 35) of Sub B Ad were heterogeneous. In order to distinguish Ad serotype 3 (Ad 3) and Ad 7 from the other serotypes of Sub B Ad, and to differentiate Ad 3 and 7 from each other, 3 different downstream primers were designed based on the sequence heterogeneity. By a single-tube PCR method using a combination of 6 primers including the 3 new primers, Ads demonstrated to amplify 188, 206, 284, and 301 bp DNA fragments for Ad 3, Ad 7, other Sub B Ads, and non-Sub B Ads, respectively. A total of 114 clinical samples were selected to evaluate the direct applicability of our PCR. The results were compared with previous culture results. Sixty-seven out of 71 (94%) Sub B Ad culture-positive samples, and 15 out of 19 (79%) Sub C or E-positive samples amplified products of the expected size. Two of 20 (10%) culture-negative samples from pharyngoconjunctival fever patients were identified as Ad 3 by the PCR. Four samples, from which non-Ad viruses were isolated, were negative by the PCR. The present study might provide a rapid and sensitive diagnosis method for infections caused by Sub B Ads.
Fujimoto
T
T
Division of Microbiology, Hyogo Prefectural Institute of Public Health, Kobe, Japan. fujimo@iph.pref.hyogo.jp
Chikahira
M
M
Kase
T
T
Morikawa
S
S
Okafuji
T
T
Yokota
Y
Y
Nishio
O
O
D000258:Adenovirus Infections, Human / Q000175:diagnosis* / Q000821:virology; D000260:Adenoviruses, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009305:Nasopharynx / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012703:Serotyping; D014776:Virus Cultivation
D036022:Capsid Proteins; D004279:DNA, Viral; C018315:hexon capsid protein, Adenovirus