Updating a New Semi-nested PCR Primer Pair for the Specific Detection of GII Norovirus in Oysters

Abstract

Oysters are major transmission vectors of noroviruses (NoVs) in the environment. Outbreaks of NoVs are often associated with the consumption of NoV-contaminated oysters. Laboratory confirmation of suspected oyster samples is a critical step in the surveillance and control of NoVs. Because of non-specific amplification, false-positive results are frequently obtained by semi-nested RT-PCR with the presently widely used primer set (G2SKF/G2SKR). Here, a novel universal PCR primer set N (NG2OF/NG2OR) specific for genogroup II (GII) NoVs was designed based on all GII NoV sequences available in public databases. Specific products were obtained with the primer set N when the NoV-positive oysters, spiked with each of five representative genotypes of GII NoVs (GII.17, GII.13, GII.4, GII.3, and GII.12), were subjected to analyzing. No products were detected with the primer set N for the NoV-negative oysters, while the primer set C gave various non-specific bands. Twenty-three out of 156 fresh oyster samples were NoV-positive with both the primer set N and the classic primer set, while eight were NoV-positive solely with the primer set N. Compared with the classic primer set, the newly designed primer set N had a higher detection rate and improved specificity for GII NoVs in oyster samples. These results show that the novel PCR primer pair is specific and applicable for the detection of GII NoVs in oysters. Supplementary Information The online version contains supplementary material available at 10.1007/s12560-022-09511-6.

Detection of novel coronaviruses from dusky fruit bat (Penthetor lucasi) in Sarawak, Malaysian Borneo

“…The positive samples of C. brachyotis were detected in guano samples and rectal swabs collected from Singapore Zoological Garden and Sungei Buloh Wetland Reserve. Although they used a nested PCR protocol (Drosten et al., 2003), their positivity rate might be low due to their primers sensitivity, unlike our study used the degenerate primers and semi‐nested PCR protocol and it is known that this protocol has a higher sensitivity rate and specificity in individual testing (Dong et al., 2022; Thi Nguyen et al., 2022).…”

Benchmarking Bioinformatic Tools for Amplicon-Based Sequencing of Norovirus

“…It has been confirmed in numerous studies that PCR cycling number and polymerase fidelity can affect the quality of DNA obtained ( 39 – 43 ). In addition to this, primer-bias can result in preferential amplification of certain genotypes, skewing the relative abundance observed following bioinformatic analysis ( 44 – 46 ). Considering the applicability of this pipeline in a food outbreak setting, attempting to link food, environmental, and patient sequences, then phylogenetic measurements such as UniFrac rank higher than rank-based methods such as Bray-Curtis.…”

Development of an amplicon-based high throughput sequencing method for genotypic characterisation of norovirus in oysters

“…We hypothesise that updated primer sets would permit the detection of additional genotypes, particularly for norovirus GII.17, GII.3 and GI.3 (4, 21, 28, 70). While a confusion matrix was used to assess method performance, an inter-laboratory ring trial would provide a more realistic measure of method performance.…”

“…Thus, designing suitable primers to capture norovirus’s existing and potential diversity is challenging. Various primers have been used for molecular characterisation, with most reference laboratories targeting the most conserved region of the genome (ORF1-ORF2 junction) and using degenerate primers that can tolerate sequence mismatches (25, 28). The regions typically targeted for molecular characterisation result in amplicons ranging from 113 bp to 587 bp in length (29).…”