Detection of encephalomyocarditis virus in clinical samples by immunomagnetic separation and one-step RT-PCR.
A method of immunomagnetic separation and one-step reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Encephalomyocarditis virus (EMCV). EMCV was captured from sample on magnetic beads with homologous monoclonal antibody and then heat denatured. The heated beads were used directly in one-step RT-PCR reaction to amplify a 285-bp PCR fragment at the 3' end of the genomic region that encodes the viral polymerase. This method detected as little as 3.5 TCID(50) of EMCV from infected cell culture. It was shown with this method that the sensitivity of RT-PCR increased when applied for the detection of EMCV added to fecal extract. Using this protocol EMCV was detected from heart homogenate samples containing less than 100 TCID(50)/ml. The amplified product was sequenced to ensure specificity. The immunomagnetic-RT/PCR procedure described here should be useful for the rapid, specific and sensitive detection of EMCV in clinical samples. This technique is rapid, reliable and can be readily adapted to detect EMCV from other clinical samples.
Kassimi
L Bakkali
LB
Agence Francaise de Securite Sanitaire des Aliments, AFSSA-Alfort, 22 rue Pierre Curie BP 67, 94703 cedex, Maisons-Alfort, France. l.bakkali@afssa.fr
Gonzague
M
M
Boutrouille
A
A
Cruciere
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D018188:Cardiovirus Infections / Q000175:diagnosis / Q000662:veterinary*; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D004686:Encephalomyocarditis virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D005243:Feces / Q000821:virology; D018189:Immunomagnetic Separation / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000821:virology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D012367:RNA, Viral