Development of a loop-mediated isothermal amplification method for rapid detection of caprine arthritis-encephalitis virus proviral DNA.

ABSTRACT

A rapid detection assay based on loop-mediated isothermal amplification (LAMP) has been developed for detecting caprine arthritis-encephalitis (CAEV) proviral DNA. The LAMP assay utilized a set of five primers designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs. There was no cross-reaction with the negative control. Amplification was monitored using a Loopamp real-time turbidimeter; turbidity and the corresponding time were recorded. Amplification from CAEV-Shanxi DNA was detected as early as 17 min, with a maximum sensitivity of 0.0001 TCID(50), reached at 32 min. Sixty-eight animal blood samples were tested using AGID, PCR and LAMP assay, and the positive rates were 30.9 %, 33.8 % and 47.1 %, respectively. Whole blood can be used directly, eliminating the need for separation of PBMCs and nucleic acid extraction, reducing the overall procedure time to approximately 80 min. Therefore, the LAMP assay provides a specific and sensitive means for detecting CAEV proviral DNA in a simple, fast, and cost-effective manner and should be useful in eradication programs and epidemiological studies. Furthermore, the LAMP assay can be performed in less-well-equipped laboratories as well as in the field.