Establishment of reverse transcription loop-mediated isothermal amplification (rt-lamp) method for the detection of virulent newcastle disease virus

Abstract from research papers submitted to the Faculty of Veterinary Medicine, Universiti Malaysia Kelantan to fulfil the requirements of the course DVT 55204 – Research Project.
Newcastle Disease Virus (NDV) is a highly contagious viral pathogen that affects various bird species, primarily domestic and wild birds, including poultry such as chickens and turkeys. It belongs to the Avian Paramyxovirus serotype 1 (APMV-1) within the Paramyxoviridae family. NDV have different pathotypes, ranging from asymptomatic to virulent strains. The virulent pathotype, termed velogenic and mesogenic, poses a significant threat due to its potential to cause severe illness and high mortality rates in poultry flocks. Birds infected with the virulent strain can exhibit a range of clinical signs, including respiratory distress, nervous system disorders, diarrhoea, and a sharp decline in egg production. Current methods to diagnose the pathotype of the virulent NDV are by Mean death time (MDT), intra-cerebral pathogenicity index (ICPI), intra-venous pathogenicity index (IVPI) and PCR-sequencing to determine the presence of multiple amino acid motif at the fusion protein cleavage site. However, these methods are time-consuming and required specialized laboratory facilities. The establishment of robust and reliable diagnostic methods is paramount for detecting virulent strains of the Newcastle Disease Virus (NDV). This study was aimed to establish Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) to detect pathogenic ND virus The results were further confirmed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and PCR sequencing to determine the presence of polybasic amino acid sequences at the F protein cleavage site for virulent viruses. The RT-LAMP method offers a promising avenue for the rapid and sensitive identification of virulent NDV strains. The results obtained from this research using the archived samples in the laboratory showed that our designed RT-LAMP detected only the virulent NDV strains, in which confirmed with the RT-PCR using primers specific to virulent NDV. For further confirmation, amino acid sequencing was performed and the RT-LAMP and RT-PCR results yielded congruent findings with amino acid sequencing results. Both RT-LAMP and RT-PCR techniques exhibit high sensitivity and specificity, however, due to its simplicity and rapidity, RT-LAMP is more preferable for diagnosing virulent NDV strains especially in low resource settings. However, the RT-LAMP results need to be further confirmed by RT-PCR and amino acid sequencing as a gold standard molecular method. This is important for timely disease management and control strategies of ND infection.
Keywords : Virulent Newcastle Disease Virus (NDV), Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP), Reverse Transcription Polymerase Chain Reaction (RT-PCR)