Immunocapture and direct binding loop mediated isothermal amplification simplify molecular diagnosis of Cyprinid herpesvirus-3.
Loop mediated isothermal amplification (LAMP) assay is used for rapid diagnosis of Cyprinid herpesvirus-3, formerly designated koi herpesvirus (KHV), with comparable sensitivity to PCR. To reduce the time required for the LAMP assay, an immunocapture (IC) and direct binding (DB) techniques were developed to exclude the DNA extraction step in molecular diagnostic procedures of the virus. Both techniques were evaluated by using PCR and CyHV-3-LAMP assays. The DB-LAMP/PCR assays were more sensitive (detecting 0.1 virus particles/ml) than the IC-LAMP/PCR assays (detecting 1 virus particle/ml). By using the SYBR Green I stain and the DB/LAMP assay the complete CyHV-3 diagnostic process can be achieved within 90 min compared to more than 5 h for the routine PCR assay. Both assays (IC/DB) could amplify successfully CyHV-3 from clinical samples which prove its application to diagnostic tests. The DB-LAMP assay is a simple, rapid, sensitive technique and applicable to the diagnosis of CyHV-3 in the field.
Soliman
Hatem
H
Fish Medicine and Livestock Management, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.
El-Matbouli
Mansour
M
D000818:Animals; D017756:Aquaculture; D002347:Carps / Q000821:virology*; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D006564:Herpesviridae / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors