Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction.
Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI-GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5 x 10(7) to 2.5 x 10(1) copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses.
Oka
Tomoichiro
T
Department of Virology II, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo, Japan. oka-t@nih.go.jp
Katayama
Kazuhiko
K
Hansman
Grant S
GS
Kageyama
Tsutomu
T
Ogawa
Satoko
S
Wu
Fang-Tzy
FT
White
Peter A
PA
Takeda
Naokazu
N
D001315:Australia; D001483:Base Sequence; D017250:Caliciviridae Infections / Q000175:diagnosis*; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000175:diagnosis*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016366:Open Reading Frames / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D029341:Sapovirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D015345:Oligonucleotide Probes