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

Abstract

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.

Detection of Enteric Viruses in Fecal Specimens from Nonbacterial Foodborne Gastroenteritis Outbreaks in Tokyo, Japan between 1966 and 1983

“…Real-time PCR was carried out using the ABI PRISM 7900HT Sequence Detection System (Thermo Fisher Scientific) with the following reaction conditions: a 10-min denaturation step at 969 C, followed by 45 cycles at 969 C for 15 s and 569 C for 1 min. The presence of SaV, RVA, RVC, AstV, and AdV, was assessed in all samples by real-time PCR as described previously (19)(20)(21). For real-time PCR assays, 5 mL of cDNA was used for SaV, RVA, RVC, and AstV, while 5 mL of nucleic acid was used for AdV.…”

Detection and genotyping of human adenovirus and sapovirus in children with acute gastroenteritis in Belém, Pará, between 1990 and 1992: first detection of GI.7 and GV.2 sapoviruses in Brazil

“…Additionally, nested PCR was performed with primers SV-F22 (5′-SMW AWT AGT GTT TGA RAT G-3′) and SV-R2 (5′-GWG GGR TCA ACM CCW GGT GG-3′), with an amplicon of 420bp 17 . Finally, quantitative polymerase chain reaction (qPCR) was performed with the Super Script III Platinum One-Step Quantitative RT-PCR System kit (Qiagen, Hilden, Germany) for the detection of all SaV genogroups within the same reaction 18 .…”

Determination of Human Sapovirus Genotypes Causing Gastroenteritis in Children under Five Years in Baghdad

“…Sets of primers and two probes were SaV124F, SaV5F, SaV1245R, SaV5TP and SaV124TP [2], targeting the highly conserved region to detect SaVs Genogroups. Amplification and detection of all SaVs Geno groups was performed using Go Taq one step RT-qPCR from (Qiagen, German) in 7500 Fast Real time PCR (Applied Biosystem).Program conditions were revers transcription at 42•C for 20 min then hot start inactivation at 95•C for 10 min followed by 45 cycles of denaturation at 94•C for 15 sec, annealing and extension at 61•C for 1 min then final extension at 72•C for 10 min.…”

“…Norovirus genogroups (GI, GII and GIV), and Sapovirus (SaVs) classified into 15 genogroups (G) based on available virus Identification Pipeline major capsid protein (VP1) sequences [2] The (GI, GII, GIV, and GV) infect humans and causes gastroenteritis outbreaks [3;4]. SaV may cause mild gastroenteritis in young children [5] Sapovirus is a non-enveloped virus, with icosahedral geometries, and T=3 symmetry it is around 27-40 nm, non-segmented linear genome sized 8.3kb in length [6] and is slightly different from that of the Norovirus (NoV) genome.…”