Construction of an internal RT-PCR standard control for the detection of human caliciviruses in stool

RT-PCR is the most sensitive assay for the detection of human caliciviruses (HuCV) in stool and environmental samples. However, false negative results are commonly obtained due to the presence of RT-PCR inhibitors. In order to exclude such false negative results, an internal control (IC) was developed for the assay by cloning a 319 nt sequence of the Norwalk virus (NV) polymerase containing a 156 nt cDNA insert. The RT-PCR assay was carried out using RNA derived from the constructed plasmid and a primer set previously described for calicivirus detection, resulting in a 475 nt product. Distinct bands of the internal control and the viral specific RT-PCR products (319 nt) were obtained when the internal control was added to the samples. Similar results were also obtained when both the control RNA and viral RNA were seeded into stool samples from asymptomatic volunteers, or when the internal control was included into positive samples. Since the primer set used in the assays can detect a wide range of strains in both norovirus and sapovirus genera, this internal control should have a broad application for the diagnosis of human caliciviruses diagnosis in both clinical and environmental samples.