|
|
|
| Fluorescent reagent in detection of porcine reproductive and respiratory syndrome virus by loop-mediated isothermal amplification (LAMP) |
| 1, 1, 1, 1, 1, 1, |
|
|
|
|
Abstract Abstract: Here we report a novel assay method of porcine reproductive and respiratory syndrome virus (PRRSV) termed reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), using fluorescent reagent as the LAMP product judgment. With one set of special primers targeting sequence containing the M gene and N gene together, the LAMP-based assay can be completed within 30 min at 63℃. The detect limit of the assay is 1000 copies per reaction determined by using a recombined plasmid containing the target sequence, which is the same as RT-PCR assay. The specificities of the primers were confirmed using genomes of various viruses. In the fluorescent reagent test, 0.05mM calcein and 0.06mM manganese were confirmed to be appropriate in LAMP product judgment, and the result was the same as turbidity test. Furthermore, RT-LAMP assays were applied to the rapid detection of PRRSV in blood samples from 103 field piglets, 56 and 48 piglets, respectively, were positive by the RT-LAMP and RT-PCR. These results suggest that the RT-LAMP assay with fluorescent reagent provides a useful tool for the diagnosis of PRRSV infections in porcine rapidly.
|
|
Received: 26 February 2009
|
|
|
|
|
|
|
