Magnetic beads-based chemiluminescent assay for ultrasensitive detection of pseudorabies virus.

ABSTRACT

A rapid, ultrasensitive and economical Pseudorabies virus (PRV) detection system based on magnetic beads (MBs) and chemiluminescence was developed in this paper. The carboxyl functionalized MBs (MBs-COOH) were covalently coupled with aminated DNA probes for capturing PRV biotinylated amplicon, the product of polymerase chain reaction (PCR). Agarose gel electrophoresis analysis approved the reliability of biotinylated amplicon. The MBs composites were incubated with alkaline phosphatase labeled streptavidin (ALP-SA) and chemiluminescene was determined by subsequently adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). The optimal conditions of the PRV detection method were 10 microM for probe concentration, 50 degrees C for hybridization temperature and 30 min for hybridization time. The limit of detection (LOD) was as low as 100 amol/5 pM of amplicon which proved that this approach for PRV detection was ultrasensitive.