Development of Enhanced Primer Sets for Detection of Norovirus

Abstract

Norovirus (NV) is a major viral pathogen that causes nonbacterial acute gastroenteritis and outbreaks of food-borne disease. The genotype of NV most frequently responsible for NV outbreaks is GII.4, which accounts for 60–80% of cases. Moreover, original and new NV variant types have been continuously emerging, and their emergence is related to the recent global increase in NV infection. In this study, we developed advanced primer sets (NKI-F/R/F2, NKII-F/R/R2) for the detection of NV, including the variant types. The new primer sets were compared with conventional primer sets (GI-F1/R1/F2, SRI-1/2/3, GII-F1/R1/F2, and SRII-1/2/3) to evaluate their efficiency when using clinical and environmental samples. Using reverse transcription polymerase chain reaction (RT-PCR) and seminested PCR, NV GI and GII were detected in 91.7% (NKI-F/R/F2), 89.3% (NKII-F/R/R2), 54.2% (GI-F1/R1/F2), 52.5% (GII-F1/R1/F2), 25.0% (SRI-1/2/3), and 32.2% (SRII-1/2/3) of clinical and environmental specimens. Therefore, our primer sets perform better than conventional primer sets in the detection of emerged types of NV and could be used in the future for epidemiological diagnosis of infection with the virus.

Genetic and epidemiological analysis of norovirus from children with gastroenteritis in Botswana, 2013–2015

“…Its symptoms include nausea, diarrhoea, vomiting, stomach cramps and fever [ 2 ]. While these symptoms can be self-limiting in healthy individuals, children, elderly and immune-compromised individuals often experience prolonged symptoms [ 3 , 4 ]. The shedding of norovirus has also been shown in asymptomatic individuals [ 5 ], and its transmission occurs mainly by the faecal-oral route, through either contaminated food or water sources [ 6 ], person to person contact or through aerosols [ 7 ].…”

Norovirus and human adenovirus occurrence and diversity in recreational water in a karst aquifer in the Yucatan Peninsula, Mexico

“…To obtain cDNA for NoV GII by RT‐qPCR the Superscript III First Strand Synthesis System (Invitrogen) was used. Nested PCR was used to separately amplify NoV GI and NoV GII using the method published by Kong et al (). The cDNA template of 5 μ l was amplified using the NoV GI forward and reverse primers, NKI‐F and NKI‐R, respectively, to obtain an expected 314 base pair (bp) size fragment of the viral capsid gene.…”

“…The cDNA template of 5 μ l was amplified using the NoV GI forward and reverse primers, NKI‐F and NKI‐R, respectively, to obtain an expected 314 base pair (bp) size fragment of the viral capsid gene. For the second PCR round, a 2 μ l aliquot from the first PCR was used as the template to amplify an expected size fragment of 305 bp using the forward and reverse primers, NKI‐F2 and NKI‐R respectively (Kong et al ). For NoV GII first round, 5 μ l of the cDNA template were used to amplify a 295‐bp fragment of the viral RNA‐dependent RNA polymerase gene with forward and reverse primers, NKII‐F and NKII‐R respectively.…”

“…For NoV GII first round, 5 μ l of the cDNA template were used to amplify a 295‐bp fragment of the viral RNA‐dependent RNA polymerase gene with forward and reverse primers, NKII‐F and NKII‐R respectively. For the second PCR reaction, 2 μ l obtained from the first PCR was used as a template to amplify a 269‐bp fragment with the forward and reverse primers, NKII‐F and NKII‐R2 respectively (Kong et al ). The cycling conditions were as described by Kong et al ().…”

“…For the second PCR reaction, 2 μ l obtained from the first PCR was used as a template to amplify a 269‐bp fragment with the forward and reverse primers, NKII‐F and NKII‐R2 respectively (Kong et al ). The cycling conditions were as described by Kong et al ().…”