title: Development of an immunomagnetic capture reverse transcription-PCR assay for the detection of Norwalk virus.
abstract: Norwalk virus (NV) is the prototype human virus of the family Caliciviridae. A rapid immunomagnetic capture/reverse transcription-(IMC/RT-)PCR assay was developed for the detection of NV. Immunomagnetic capture (IMC) utilizes paramagnetic beads coupled to a virus-specific antibody and allows separation of virus from contaminating materials and virus concentration in a single step. The detection limit of the developed assay was approximately 250-750 genomic equivalents/ml of 10% stool suspension. The detection limit of the assay was not altered by the presence of excess hepatitis A virus (HAV), although non-specific binding of HAV to the paramagnetic beads was observed. A panel of 100 stools from experimental human infections was screened for NV using a previously described heat release method, an antigen ELISA, or IMC/RT-PCR. NV was detected in 65/100 of these samples by IMC/RT-PCR compared to only 38/99 by heat release and 31/95 by antigen detection ELISA. All samples that were negative by IMC were also negative by both heat release and antigen ELISA. The number of samples in which RT-PCR was inhibited was greatly reduced by the use of IMC/RT-PCR compared to the heat release method (1/100 and 16/95 samples inhibited, respectively). The ability of IMC to concentrate virus (> or =2000-fold greater than heat release) and effectively remove inhibitory substances gives this assay distinct advantages over both the heat release and antigen ELISAs.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Mail Stop BCM-385, 1 Baylor Plaza, Houston, TX, 77030, USA.
authors.forename: S G
authors.initials: SG
authors.lastname: Gilpatrick
authors.forename: K J
authors.initials: KJ
authors.lastname: Schwab
authors.forename: M K
authors.initials: MK
authors.lastname: Estes
authors.forename: R L
authors.initials: RL
authors.lastname: Atmar
mesh_terms: D017250:Caliciviridae Infections / Q000821:virology; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology; D006801:Humans; D018189:Immunomagnetic Separation; D009663:Norwalk virus / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity