Novel Light-Upon-Extension Real-Time PCR Assay for Simultaneous Detection, Quantification, and Genogrouping of Group A Rotavirus

Abstract

We have developed a light-upon-extension (LUX) real-time PCR assay for detection, quantification, and genogrouping of group A rotavirus (RV), the most common cause of acute gastroenteritis in children. The LUX system uses a fluorophore attached to one primer and having a self-quenching hairpin structure, making it cost-effective and specific. We designed genogroup-specific primers having different fluorophores, making it possible to differentiate between the two main genogroups of human group A RVs. The assay was applied on clinical stool specimens from Sweden and Central America (n ‫؍‬ 196) and compared to immunological and conventional PCR assays. The genogrouping ability was further validated against a subset of clinical specimens, which had been genogrouped using monoclonal antibodies. Our real-time PCR assay detected and quantified all positive specimens (n ‫؍‬ 145) and exhibited higher sensitivity than immunological assays and conventional PCR. The assay exhibited a wide dynamic range, detecting from 5 to >10 7 genes per PCR, resulting in a theoretical lower detection limit of <10,000 viruses per gram of stool. No cross-reaction was observed with specimens containing norovirus, sapovirus, astrovirus, or adenovirus. In total, 22 (15%) of the positive clinical specimens were identified as genogroup I, 122 (84%) were identified as genogroup II, and 1 specimen was found to contain a mix of both genogroups. All genogroup I-positive specimens were associated with capsid glycoprotein 2 (G2). No significant difference in viral load was found between genogroups or geographic region. The detection and quantification, combined with the genogrouping ability, make this assay a valuable tool both for diagnostics and for molecular epidemiological investigations.

Vaccine preventable viral diseases and risks associated with waterborne transmission

“…This study was conducted on severely affected children, and highlights the chance for large amounts of infectious virus to be released into sewage, and possibly enter the environment and food routes of spread and transmission. Several other reports during decades of rotavirus investigation confirm this assumption, also using modern molecular detection approaches [72].…”

Bioluminescent Enzyme Immunoassay for the Detection of Norovirus Capsid Antigen

“…In the dilution test with NV specimens, the analytical sensitivities of the BLEIA were confirmed to be 10 5 and 10 6 copies/g of fecal sample. According to the previous studies, various real-time RT-PCRs have the highest sensitivity, ranging from 10 4 to 10 5 copies/g of fecal sample, but they are not easy to perform and are timeconsuming (11,22). On the contrary, although several commercially available molecular methods have a little less sensitivity, ranging from 10 5 to 10 6 copies/g of fecal sample, they are more convenient and have also been widely used as a highly sensitive detection method for NV (7,9,21).…”

Rotavirus in diarrheal children in rural Burkina Faso: High prevalence of genotype G6P[6]

“…All rotavirus positive specimens were quantified and VP6 subgrouped by the use of a LUX real-time PCR as described previously (Nordgren et al, 2010). This real-time PCR uses labeled primers with different fluorophores for each VP6 subgroup and external plasmid standards for semi-quantification (Nordgren et al, 2010).…”