Comparison of Loop-Mediated Isothermal Amplification and PCR for the Detection and Differentiation of Marek's Disease Virus Serotypes 1, 2, and 3

Abstract

The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek's disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulent MDV and turkey herpesvirus where SYBR Green was added to the reaction mixture, whereas the SB-1-positive samples presented orange illumination after GelRed staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1-2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers.