Rapid detection of Marek's disease virus in feather follicles by loop-mediated amplification

Marek's disease (MD) remains a serious problem in the production of poultry. The disease is caused by Marek's disease virus (MDV), and despite the ubiquitous use of vaccination to control losses, MD still affects poultry farming worldwide. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) method for the simple and inexpensive detection of MDV in feather tips of chickens. Two pairs of specific primers complementary to the meq oncogene of MDV were designed, targeting the sequence of the very virulent MDV strain, RB1B. Bst polymerase was used for the isothermal amplification of viral DNA at 65 C for 90 min in a water bath. The fluorescence signal was identified in MDV-positive samples after the addition of SYBR Green and ultraviolet (UV) illumination. The sensitivity of LAMP was 2 log 10 plaque-forming units (PFU)/ml of HPRS16 and 10(3) copies/il of plasmid containing the target gene (meq) and was equal in sensitivity to PCR amplification. Due to the use of three sets of primers, LAMP was highly specific for MDV-1 DNA. The developed LAMP technique is a rapid and simple tool for the specific detection of MDV in samples of feathers taken from live chickens. Since the use of thermocyclers is not necessary for LAMP assay, it can be conducted by small laboratories and even field veterinarians.