Development of an immunomagnetic capture reverse transcription-PCR assay for the detection of Norwalk virus

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 (greater than or equal to 2000-fold greater than heat release) and effectively remove inhibitory substances gives this assay distinct advantages over both the heat release and antigen ELISAs. (C) 2000 Elsevier Science B.V. All rights reserved.