Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction

• Published in: Journal of medical virology 2006-10, Vol.78 (10), p.1347-1353
• Main Authors: Oka, Tomoichiro, Katayama, Kazuhiko, Hansman, Grant S., Kageyama, Tsutomu, Ogawa, Satoko, Wu, Fang-Tzy, White, Peter A., Takeda, Naokazu
• Format: Article
• Language: English
• Subjects: Adenovirus; Astrovirus; Australia; Base Sequence; Biological and medical sciences; Caliciviridae Infections - diagnosis; Feces - virology; Fundamental and applied biological sciences. Psychology; Gastroenteritis - diagnosis; Genome, Viral; Human viral diseases; Infectious diseases; Medical sciences; Microbiology; Miscellaneous; Molecular Sequence Data; Norovirus; Oligonucleotide Probes; Open Reading Frames - genetics; polymerase-capsid junction; real-time RT-PCR; Reverse Transcriptase Polymerase Chain Reaction - methods; Rotavirus; sapovirus; Sapovirus - genetics; Sapovirus - isolation & purification; Sensitivity and Specificity; Sequence Alignment; TaqMan MGB probe; Viral diseases; Virology
• ISSN: 0146-6615; 1096-9071
• DOI: 10.1002/jmv.20699

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 × 107 to 2.5 × 101 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. J. Med. Virol. 78:1347–1353, 2006. © 2006 Wiley‐Liss, Inc.