Detection Of Human Caliciviruses Research Articles

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.

I Congreso Nacional (segunda reunión) de la Rama de Bioquímica y Biología Molecular de Virus de la Sociedad Mexicana de Bioquímica (Segunda Parte)

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Incidence of human calicivirus and rotavirus infection in patients with gastroenteritis in South Africa.

Human caliciviruses (HuCVs) are reportedly responsible for 2.5-4% of nonbacterial sporadic gastroenteritis. The incidence of HuCV infection in South Africa is unknown. Stool specimens from 1,296 South African patients with sporadic gastroenteritis were screened for the presence of HuCVs using electron microscopy, recombinant enzyme immunoassays for Norwalk (NV) and Mexican (MX) viruses, and the reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR products were sequenced to ascertain which HuCV genogroups were present. HuCVs were detected in 43/1,296 (3.3%) specimens examined, with RT-PCR proving to be the most sensitive detection method. Genetic analysis of the isolates indicated that 81% were Snow Mountain Agent, or MX-like; 8% were NV-like; and 11% were HuCV/Sapporo-like. This study indicates that a combination of assays is needed for the accurate detection of HuCVs. Comparative data on hospitalised patients showed that the incidence of rotavirus infection was approximately ten times greater than that of HuCV infection.