Development Of An Antigen Capture Reverse Transcriptase/Polymerase Chain Reaction For Detection Foot-and-Mouth Disease Virus

ABSTRACT FMDV was proliferated from the culture medium of BHK21, then FMDV146S is extracted by polyethylene glycol-6000 (PEG-6000) precipitation, differential centrifugation, ultra-centrifugation and sucrose density gradient. Vaccinate rabbit using prepared antigen,and separate sera when titre arrived to demand quality. Type-specific IgG was prepared through (NH4)2SO4, sephadex G-25 and DEAE-cellulose. Then capture FMDV from clinical material with sterilized Eppendrof tubes of coating type-specific IgG, Amplify the conserved viral sequences by common primers (FM1/FM4). Therefore, we developed a method of antigen capture reverses transcriptase /polymerase chain reaction. Meantime, after the test such as coating antibody, capturing antigen, storage period of coated antibody, sensitivity and specificity, field samples, the results shown that the efficiency of antibody coating and antigen captured were improved by glutarldehyde fixation and BSA saturation. Antibody is still able to capture antigen successfully after storage for 1 month or overnight at room temperature. The result has also shown that compared to regular RT/PCR, AC-RT/PCR has the same sensitivity that can examine virus with 0.1 LD50, and higher specificity than regular RT/PCR. Besides, since the assay use one step RT/PCR, the whole process of AC-RT/PCR is completed in one Eppendorf tube and free from indirect contamination, which simplified the operation. Also AC-RT/PCR may examine a number of samples in test.